Embryonic Callus Induction Research Articles

Population genomic analysis reveals key genetic variations and the driving force for embryonic callus induction capability in maize

Genetic transformation has been an effective technology for improving the agronomic traits of maize. However, it is highly reliant on the use of embryonic callus (EC) and shows a serious genotype dependence. In this study, we performed genomic sequencing for 80 core maize germplasms and constructed a high-density genomic variation map using our newly developed pipeline (MQ2Gpipe). Based on the induction rate of EC (REC), these inbred lines were categorized into three subpopulations. The low-REC germplasms displayed more abundant genetic diversity than the high-REC germplasms. By integrating a genome-wide selective signature screen and region-based association analysis, we revealed 95.23 Mb of selective regions and 43 REC-associated variants. These variants had phenotypic variance explained values ranging between 21.46 and 49.46%. In total, 103 candidate genes were identified within the linkage disequilibrium regions of these REC-associated loci. These genes mainly participate in regulation of the cell cycle, regulation of cytokinesis, and other functions, among which MYB15 and EMB2745 were located within the previously reported QTL for EC induction. Numerous leaf area-associated variants with large effects were closely linked to several REC-related loci, implying a potential synergistic selection of REC and leaf size during modern maize breeding.

Optimization of Key Technologies for Induction of Embryogenic Callus and Maturation of Somatic Embryos in Korean Pine (Pinus koraiensis)

Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low induction and proliferation rates, hindering the production of improved plant materials. We investigated the effects of the explant sterilization method, 4 °C cryopreservation, basal medium, ethylene removal, liquid medium supplementation, and a combination of PGRs on embryogenic callus (EC) induction of Korean pine, using immature embryos of Korean pine as explants. The effects of sucrose and maltose on EC proliferation and maturation were investigated. The differences in the maturation ability of EC somatic embryos before and after cryopreservation were evaluated using the induced embryonic cell lines. The results showed that zygotic embryos (ZEs) performed better than megagametophytes (MGs) as explants. The induction rate of EC was significantly increased after 28 days of cryopreservation at 4 °C. The induction rate of EC in the #5 family increased from 10.00% to 62.8%. The EC induction rate of the five families cultured with the DCR basal medium was higher than that with the mLV basal medium. Among them, the induction rate of the #5 family cultured with the mLV basal medium was 23.3%, while that with the DCR basal medium was 60.9%, an increase of 2.6 times. There was no significant difference in the maturation ability of EC somatic embryos before and after cryopreservation. In conclusion, this study provides a method to improve the EC induction rate and maturation ability of Korean pine.

GWAS across multiple environments and WGCNA suggest the involvement of ZmARF23 in embryonic callus induction from immature maize embryos.

Combined GWAS, WGCNA, and gene-based association studies identified the co-expression network and hub genes for maize EC induction. ZmARF23 bound to ZmSAUR15 promoter and regulated its expression, affecting EC induction. Embryonic callus (EC) induction in immature maize embryos shows high genotype dependence, which limits the application of genetic transformation in transgenic breeding and gene function elucidation in maize. Herein, we conducted a genome-wide association mapping (GWAS) for four EC induction-related traits, namely rate of embryonic callus induction (REC), increased callus diameter (ICD), ratio of shoot formation (RSF), and length of shoot (LS) across different environments. A total of 77 SNPs were significantly associated these traits under three environments and using the averages (across environments). Among these significant SNPs, five were simultaneously detected under multiple environments and 11 had respective phenotypic variation explained > 10%. A total of 257 genes were located in the linkage disequilibrium decay of these REC- and ICD-associated SNPs, of which 178 were responsive to EC induction. According to the expression values of the 178 genes, we performed a weighted gene co-expression network analysis (WGCNA) and revealed an EC induction-associated module and five hub genes. Hub gene-based association studies uncovered that the intragenic variations in GRMZM2G105473 and ZmARF23 influenced EC induction efficiency among different maize lines. Dual-luciferase reporter assay indicated that ZmARF23 bound to the promoter of a known causal gene (ZmSAUR15) for EC induction and positively regulated its expression on the transcription level. Our study will deepen the understanding of genetic and molecular mechanisms underlying EC induction and contribute to the use of genetic transformation in maize.

Establishment of highly efficient plant regeneration of Paeonia ostii ‘Fengdan’ through optimization of callus, adventitious shoot, and rooting induction

Tree peony is a famous ornamental plant, while the low propagation rate is the main hurdles hindering the industry development. Till now, the highly efficient regeneration system for tree peony is not established. In this study, using Paeonia ostii ‘Fengdan’ mature embryos, the effects of variations in inoculation method, initiating culture, adventitious shoot induction, rooting media, plant growth regulators (PGRs), and a non-conventional PGR (plant extracts) on regeneration from explants were evaluated. In embryo cultures, embryonic callus induction rate of 1/4 embryos was the highest among those of embryos with other three technical treatments (whole embryos, 1/2 embryos, and pieces of embryos). The woody plant medium (WPM) containing 1.0 mg • L − 1 6-BA, 0.5 mg • L − 1 GA3, 30.0 g • L − 1 sucrose, and 3.0 g • L − 1 phytagel significantly improved shoot induction and multiplication. 3.0 mg • L − 1 plant extracts promoted hypocotyl germination, rooting, and root growth, in direct embryo culture, and a combination of 3.0 mg • L − 1 plant extracts + 2.0 mg • L − 1 IBA + 1.5 mg • L − 1 IAA produced optimal rooting induction rate for multiple shoots in direct embryo culture and indirect somatic embryogenesis. For the three in vitro micropropagation methods, the highest shoot proliferation coefficient (5.4 ± 0.2) was obtained with indirect somatic embryogenesis. Fortunately, the propagation ability of shoots remains high, even when culture propagation was continued for more than two years. Thus, a reliable system for plant regeneration from mature embryos derived from P. ostii ‘Fengdan’ callus and two direct embryo culture systems have been established. The novel regeneration system could facilitate uniform seedling production.

A High-Quality Reference Genome Sequence and Genetic Transformation System of Aralia elata.

Aralia elata is a perennial woody plant of the genus Aralia in the family Araliaceae. It is rich in saponins and therefore has a wide range of pharmacological effects. Here, we report a high-quality reference genome of A. elata, with a genome size of 1.21 Gb and a contig N50 of 51.34 Mb, produced by PacBio HiFi sequencing technology. This is the first genome assembly for the genus Aralia. Through genome evolutionary analysis, we explored the phylogeny and whole genome duplication (WGD) events in the A. elata genome. The results indicated that a recent WGD event occurred in the A. elata genome. Estimation of the divergence times indicated that the WGD may be shared by Araliaceae. By analyzing the genome sequence of A. elata and combining the transcriptome data from three tissues, we discovered important genes related to triterpene saponins biosynthesis. Furthermore, based on the embryonic callus induction system of A. elata established in our laboratory, we set up the genetic transformation system of this plant. The genomic resources and genetic transformation system obtained in this study provide insights into A. elata and lays the foundation for further exploration of the A. elata regulatory mechanism.

Genome assembly of the maize inbred line A188 provides a new reference genome for functional genomics

The current assembled maize genomes cannot represent the broad genetic diversity of maize germplasms. Acquiring more genome sequences is critical for constructing a pan-genome and elucidating the linkage between genotype and phenotype in maize. Here we describe the genome sequence and annotation of A188, a maize inbred line with high phenotypic variation relative to other lines, acquired by single-molecule sequencing and optical genome mapping. We assembled a 2210-Mb genome with a scaffold N50 size of 11.61 million bases (Mb), compared to 9.73 Mb for B73 and 10.2 Mb for Mo17. Based on the B73_RefGen_V4 genome, 295 scaffolds (2084.35 Mb, 94.30% of the final genome assembly) were anchored and oriented on ten chromosomes. Comparative analysis revealed that ~30% of the predicted A188 genes showed large structural divergence from B73, Mo17, and W22 genomes, which causes high protein divergence and may lead to phenotypic variation among the four inbred lines. As a line with high embryonic callus (EC) induction capacity, A188 provides a convenient tool for elucidating the molecular mechanism underlying the formation of EC in maize. Combining our new A188 genome with previously reported QTL and RNA sequencing data revealed eight genes with large structural variation and two differentially expressed genes playing potential roles in maize EC induction.

INDIRECT SOMATIC EMBRYOGENESIS ON MUTANTS OF Agave tequilana WEBER CULTIVAR BLUE INDUCED WITH CO60 GAMMA RAYS

<p><strong>Background:</strong> Blue agave (<em>Agave tequilana </em>Weber var. azul)<strong>,</strong> it is commonly known as “Agave azul or agave tequilero”. The Agave crop is fundamental part of the productive chain Agave-Tequila, since is the distinctive source of reducing sugars for the elaboration of the liqueur known as Tequila, according with the Appellation of Origen Tequila to fulfill the National Normativity (NOM-). Besides, Tequila is the most spirit drink exported from Mexico worldwide. According with the latest evaluation over this crop, the susceptibility to pathogens generate in cases total lost of the agave fields. <strong>Objective.</strong> to achieve somatic embryos that could be resistant, generated from plantlets irradiated with Co<sup>60</sup>gamma rays; which were obtained from axillary buds. <strong>Methodology:</strong> The arisen of embryo was reached in the modified Murashige and Skoog (MS) medium supplemented with of 2,4-D combined with BA or KIN. The plantlets were radiated 12 weeks after the somatic embryo induction with Co<sup>60</sup> gamma rays. The doses were 0 (test control), 10, 20 and 30 Gy. <strong>Results:</strong> There was a significant difference in the induction of embryonic callus, development and mature somatic embryos, which were reduced as the doses of radiation increases. Since only somatic embryos were achieved in tissues treated with 10 and 20 Gy doses, in higher doses induction of embryonic callus was inhibited. The regression confirmed the negative results with the increase of radiation. <strong>Implications:</strong> There were made protocols to achieve somatic embryos and plants from tissues irradiated with 10 and 20 Gy. <strong>Conclusions</strong>: The radiation effect over somatic tissue was crucial as the doses affect the conversion into plantlets, nevertheless such radiation allowed the conversion of the embryos to complete agave plantlets</p>

The regeneration of Acer rubrum L. \u201cOctober Glory\u201d through embryonic callus

BackgroundTissue culture and rapid propagation technology is an important way to solve the difficulties of plant propagation. This experiment aims to explore the appropriate conditions at each stage of the red maple’s tissue culture process and to obtain plantlets, thus providing a theoretical basis for the establishment of the red maple’s tissue culture system.ResultsThe results showed that the stem segment is the most suitable explant for inducing embryogenic callus. The MS (Murashige&Skoog) + 0.8 mg/L TDZ (Thidiazuron) + 1.0 mg/L 6-BA (6-Benzylaminopurine) + 0.5 mg/L IAA(Indole-3-acetic acid) + 35 g/L sucrose+ 7.5 g/L semi-fixed medium was the best for callus formation. When selecting type VI callus as embryonic callus induction material, MS + 0.6 mg/L TDZ + 0.5 mg/L 6-BA + 2.0 mg/L IAA + 35 g/L sucrose+ 7.5 g/L semi-fixed medium can get embryonic callus. The optimal medium for adventitious bud induction is MS + 1.0 mg/L TDZ + 3.0 mg/L 6-BA+ 0.2 mg/L NAA (1-Naphthaleneacetic acid) + 1.2 mg/L IAA + 35 g/L sucrose+ 7.5 g/L semi-fixed medium. The induction rate of adventitious roots in MS + 0.6 mg/L TDZ + 1.0 mg/L 6-BA+ 3 mg/L NAA + 35 g/L sucrose+ 7.5 g/L semi-fixed medium was the highest, reaching 76%.ConclusionsIn the course of our research, we found that PGRs play an important role in the callus induction stage, and the effect of TDZ is particularly obvious; The callus cells grow and proliferate according to the “S” growth curve, and can be sub-cultured when the highest growth point is reached to maintain the rapid proliferation of the callus cells and to avoid inactivation of callus caused by tight niche.

Somatic embryo induction and Agrobacterium-mediated transformation of embryonic callus tissue in Phoebe bournei, an endangered woody species in Lauraceae

Phoebe bournei, a plant species endemic to China, is a precious timber tree and widely used in landscaping. This tree contains numerous secondary metabolites, underscoring its potential economic value. However, studies on this species, including molecular genetic research, remain limited. In this study, both a somatic embryogenesis (SE) technical system and Agrobacterium-mediated genetic transformation were successfully employed in P. bournei for the first time. The SE technical system was constructed using immature embryos as original material. The primary embryo and embryonic callus induction rates were 30.66% and 41.67%, respectively. The highest rate of embryonic callus proliferation was 3.84. The maximum maturity coefficient and germination rate were 53.44/g and 39%, respectively. Agrobacterium-mediated genetic transformation was performed using the SE technical system, and the highest transformation rate was 11.24%. The results presented here are the first to demonstrate an efficient approach to achieve numerous P. bournei plantlets, which serves as the basis for artificial cultivation and resource conservation. Furthermore, the genetic transformation platform constructed in this study will facilitate assessment of gene function and molecular regulation.

Screening of factors affecting somatic callusing and embryo induction in Allium cepa L. through Plackett–Burman methodology

Induction of embryonic callus through available asexual explant culture is applied in the protection and maintenance of cytoplasmic-male sterility and in the maintenance of other lines of onion (Allium cepa L.). Usage of the Plackett?Burman (PB) experimental design has been suggested as a screening method for many variables that influence the desired induction. In order to overcome limitations of standard experimental designs and prioritization of media culture components, a systematic investigation was carried out using the PB design. In this investigation, the effects of different factors such as 2,4-D, putrescine, carbohydrate sources (maltose, sorbitol, and sucrose), silver nitrate, glutamine, and glycine concentrations were assayed on callus and embryo induction through mature zygotic embryo explant culture. Out of the eight medium components screened, the four components of putrescine, glutamine, 2,4-D, and glycine were found to contribute positively to the callus production with a maximum production rate of 97.14%. The normal plot and linear regression equation with positive largest coefficient showed that putrescine had the highest significant positive effect with 200 mg/L and three elements comprising 2,4-D and glycine showing a significant reducing effect on callus induction. In embryonic callus induction, a wide range of responses were observed from 0% to 100% in the 21 trials. Results of linear regression data showed that among the eight elements, 2,4-D, glutamine, and glycine had significant effects on embryo production. Glycine and 2,4-D had the highest (26.34%) and the lowest (8.33%) contribution to explaining embryonic callus induction, respectively. A wide range of responses (0 to 44.1 mg) were observed in the Plackett-Burman design with respect to the fresh weight. The regression analysis for the Plackett-Burman design (PBD) demonstrated that glycine, sorbitol, and sucrose were of high influence on the fresh weight (P ≤ 0.05). Glycine (31.4%) and sucrose (6.7%) were found to have the highest and lowest contribution to the fresh weight of callus, respectively. Thus, this practical article seeks to show some helpful statistical approaches to typical problems in data analyses of tissue culture research.

Establishment of a maize callus regeneration system from haploid shoot tips

For maize transgenic breeding, a major bottleneck is time need to produce homozygous transgenic plants by selfing of one or more additional generations. This study established an effective haploid maize callus regeneration system using haploid shoot tips of maize which enables to form homozygous transgenic insertions in a single generation. Haploid shoot tips were cultured for haploid embryonic callus induction, which was then transformed with a GFP-contained vector and generated transgenic haploid plants. We tested different seed sterilization methods, germination manners, shoot lengths, cutting patterns, medium components, light treatments in differentiation and particle bombardment distances. The results indicated that HgCl2 was better than H2O2 for decreasing seeds contamination rate. Seedlings with 2.0–6.0 cm in length was optimal for the high frequency of embryonic callus induction. Then, the best culture medium components were “MS + 2 mg/L 2,4-D + 1 mg/L 6-BA + 500 mg/L CH + 10 g/L Mannitol + 7 mg/L AgNO3 + 1 mg/L B5” and “MS + 1 mg/L 6-BA + 0.4 mg/L IBA + 500 mg/L CH + 1 mL/L Cephalothin”, respectively, for callus induction and regeneration. Callus with 4 mm in size and 4 generations of subculture was better than the others for particle bombardment to obtain positive transgenic plants. Among the six genotypes of haploids, haploid 18-599 W had the highest induction rate of embryonic callus. Interestingly, each of the haploids did not show different induction rate compared to its diploid, except haploid 18-599R. Our research provides new insights for rapid homozygosis of transformation events in maize. An effective haploid maize shoot tips callus regeneration system was established which enabled homozygous transgenic plants to be obtained in a single generation.

Micropropagation and Somatic Embryogenesis Induction of Gardenia jasminoides Plants

Gardenia jasminoides Ellis is an evergreen plant with fragrant creamy white flowers and glossy, darkgreen leaves, belongs to family Rubiaceae and member of the genus Gardenia. The current study aimed to afford a new technique for in vitro propagation and callus induction of G. jasminoides by using single nodes excised from soft cuttings using full strength MS salts supplemented with 30g/l sucrose, 4g/l gelrite and different concentrations of plant growth regulators. Generally, the current study showed that NAA and BA at 0.0 and 1.0 mg/l, respectively gave rise to the best results for initiation stage. Meanwhile, fortified the basal MS medium with BA and NAA at 2.0 and 0.250 mg/l, consecutively; resulted in the best results with respect to multiplication stage. Regarding rhizogenesis, the neoformed shoots produced from vegetative multiplication stage were rooted successfully upon cultured on MS medium supplemented with 1.0 mg/l IBA and NAA at either 0.250 or 0.500 mg/l, whereas the highest number of roots was recorded besides callus induction. Regarding embryonic callus induction, adding 2,4-D at 1.0 mg/l; enhanced embryonic callogenesis. Neoformed plantlets were acclimatized ex vitro and in vivo vigorously in mixture of perlite and peatmoss at (1:1, v/v), which resulted in the highest percentage value of survival / percent (100%) and successfully flowered and showed true-to-type plants ex vitro.

Integrative analysis of DNA methylation, mRNAs, and small RNAs during maize embryo dedifferentiation

BackgroundMaize (Zea mays) is an important model crop for transgenic studies. However, genetic transformation of maize requires embryonic calli derived from immature embryo, and the impact of utilizing tissue culture methods on the maize epigenome is poorly understood. Here, we generated whole-genome MeDIP-seq data examining DNA methylation in dedifferentiated and normal immature maize embryos.ResultsWe observed that most of the dedifferentiated embryos exhibited a methylation increase compared to normal embryos. Increased methylation at promoters was associated with down-regulated protein-coding gene expression; however, the correlation was not strong. Analysis of the callus and immature embryos indicated that the methylation increase was induced during induction of embryonic callus, suggesting phenotypic consequences may be caused by perturbations in genomic DNA methylation levels. The correlation between the 21-24nt small RNAs and DNA methylation regions were investigated but only a statistically significant correlation for 24nt small RNAs was observed.ConclusionsThese data extend the significance of epigenetic changes during maize embryo callus formation, and the methylation changes might explain some of the observed embryonic callus variation in callus formation.

Metabolomic and Proteomic Analysis of Maize Embryonic Callus induced from immature embryo

The low ratio of embryonic callus (EC) induction has inhibited the rapid development of maize genetic engineering. Still, little is known to explain the genotype-dependence of EC induction. Here, we performed a large-scale, quantitative analysis of the maize EC metabolome and proteome at three typical induction stages in two inbred lines with a range of EC induction capabilities. Comparison of the metabolomes and proteomes suggests that the differential molecular responses begin at an early stage of development and continue throughout the process of EC formation. The two inbred lines show different responses under various conditions, such as metal ion binding, cell enlargement, stem cell formation, meristematic activity maintenance, somatic embryogenesis, cell wall synthesis, and hormone signal transduction. Furthermore, the differences in hormone (auxin, cytokinin, gibberellin, salicylic acid, jasmonic acid, brassinosteroid and ethylene) synthesis and transduction ability could partially explain the higher EC induction ratio in the inbred line 18-599R. During EC formation, repression of the “histone deacetylase 2 and ERF transcription factors” complex in 18-599R activated the expression of downstream genes, which further promoted EC induction. Together, our data provide new insights into the molecular regulatory mechanism responsible for efficient EC induction in maize.

Embryonic Callus Induction and Plant Regeneration of Lilium fargesii

In order to rationally protect and utilize the important wild traditional Chinese medicine resources,we built an efficient and instability regeneration system of Lilium fargesii Franch. by embryonic callus induction. With the scales as explants,by an orthogonal experiment,we studied the effect of different kinds and contents of plant hormone on embryonic callus induction and plantlet regeneration,and established a propagation system by tissue culture. The optimal medium for inducing embryonic callus was MS + 6-BA 0. 5 mg·L~(-1)+NAA 0. 5 mg · L~(-1)+ 2,4-D 0. 1 mg · L~(-1),the callus induction rate was 89. 29%,and the generation coefficient of bulblet was 4. 7. The optimal medium for regeneration of embryonic callus and inducing the bulblets was MS + 6-BA 1. 0 mg · L~(-1)+ 2,4-D 0. 1 mg · L~(-1),and its regeneration rate was 5. 0 /35 d. The optimal rooting medium was 1 /2MS + NAA 0. 2 mg·L~(-1),and the rooting rate was 100%. There were 90% of plantlet surviving after transplanting rooted plantlets into sand when cultured in constant temperature and humidity condition for 35 d. Our study could provide a basis on protecting the improved trait of wild resources of L. fargesii,and lay the foundation for expanding the artificial cultivation and embryoid asexual breeding.

In vitro propagation of Santalum album L.

Santalum album L. (sandalwood) is a valuable tropical plant species that belongs to the family Santalaceae. Santalol - the active compound in S. album, which is commonly known as sandalwood oil is highly valued in the perfumery industry due to its sweet persistent aroma. Sandalwood plants are over-exploited for harvesting their wood. Although the species is naturally regenerated by seeds the success rate is as low as 20 %. Due to the hemi-parasitic nature of S. album , the survival of seedlings is low making the species rare in Sri Lanka. There is a high demand for sandalwood plants for commercial scale plantations. Therefore in the present study, plantlet regeneration through somatic embryogenesis was studied in order to produce a large number of healthy plants to be used in establishing commercial scale plantations. Mature and immature seeds, leaf discs and nodal segments were used as explants for embryonic callus induction. Nodal segments found to be the best explants for embryonic callus production. Murashige and Skoog medium (MS) supplemented with 2.5 mg/L 2,4-dichloro phenoxy acetic acid (2,4-D) and 3.0 mg/L kinetin (kin) induced callus with a mean diameter of 3.22 ± 0.1 cm after 8 weeks of incubation. Somatic embryo induction was optimized by the addition of 0.5 mg/L benzyl amino purine (BAP), 1.0 mg/L indole-3-acetic acid (IAA) and 0.5 mg/L kin to MS medium, which resulted about 10 somatic embryos per 1.0 cm2 of callus. Somatic embryos germinated best in MS medium supplemented with 2.0 mg/L gibberellic acid (GA3). The highest percentage of plantlet regeneration was observed when the germinated embryos were transferred into MS medium supplemented with 0.4 mg/L BAP and 0.2 mg/L IAA. J.Natn.Sci.Foundation Sri Lanka 2015 43 (3):265-272

Study on plant regeneration from somatic embryos of vulnerable medicinal plant Glehnia littoralis

To study the cause of the seeds dormancy of Glehnia littoralis in vitro and to establish plant regeneration methods via somatic embryos. The effects of endosperm and exogenous hormone on the seed dormancy breaking of G. littoralis and the effect of hormone concentration on embryonic callus induction and plant regeneration via somatic embryos were observed, The germination rate of the seeds with 1/3 endosperm was the highest which achieved 31%. TDZ, 6-BA and GA3 treatment could not break seed dormancy but easily lead to abnormal seedlings. Embryogenic callus induction rates was up to 57% on MS supplemented with 1.0 mg x L(-1) 2,4-D. After 20 days culture, embryogenic calli were transferred to MS medium and cotyledonary embryos were formed in 40 days. The regenerated plants were obtained in 20 days. An effective system of plant regeneration of G. littoralis was established in this study.

Establishment of high efficiency genetic transformation system of maize mediated by <I>Agrobacterium tumefaciens</I>

In order to establish high-frequency regeneration and high-efficiency genetic transformation system in maize, the significance of the 11 factors influencing maize embryonic callus induction and 9 factors affecting embryonic callus differentiation was researched by orthogonal experiment. The results showed that genotype had highly significant impact on induction of embryonic callus. The concentration of 6-BA, AgNO3, 2,4-D, ABA, and medium are the significant factors. The Multi-comparison showed that ABA 2 mg/L has a significant influence. Among the callus differentiation factors, the genotype and 6-BA concentration showed a strong main effect, the concentrations of NAA, medium, KT and 2,4-D had significant impacts on callus differentiation. Southern blotting analysis demonstrated that the resistant callus rate under the selection pressure of 25 mg/L hygromycin was a reliable indicator for system optimization in resistance screening. The concentration of acetosyringone (AS) showed sensitive differences among genotypes. The highest transformation rate was found with the optimized combination of 24-25 degrees C for co-culture temperature, 0.7 ODx15 min for Agrobacterium tumefa-ciens concentration and incubation-time, and pH 5.5-6.2. By this optimized combination, the survival rate of resistant calli as an index for the stable transformation rates of inbred lines Huangzao 4 and Zong 31 by introducing GUS gene into maize inbred lines was as high as 48.6% and 46.2%, respectively.

Analysis of Gene Effect on Four Characters of Immature Embryo Culture in Maize

This study was to find the regularity in the hereditary variation for the main culturing characters of the immature embryo culture in maize. Two kinds of inbred-line, R18-599 (red) with very excellent embryo culturing capacity and R15 with very poor embryo culturing capacity, were used as P 1 and P 2 for obtaining six generations. By culturing immature embryos of the six generations, four culturing characters, namely embryonic callus induction efficiency, nonembryonic callus induction efficiency, cloning ability of the embryonic callus, and number of regenerating plants, were analyzed using the general mean analysis and generation joint analysis. Results showed that the embryonic callus induction efficiency accorded with two major additive-dominance-epistatic genes and polygene-mixed additive-dominance-epistatic inheritance model. The induction efficiency of the nonembryonic callus accorded with two major additive-dominance-epistatic genes. The number of regenerating plants accorded with one major gene and polygene-mixed additive-dominance inheritance model. The cloning ability of the embryo callus accorded with two major genes and polygene-mixed inheritance model, whereas the effect of epistatic gene on this character was identified to be different using the two methods. By comparison of the results of the two methods, generation joint analysis may not only raise experimental precision but also provide more genetic information.

Regeneration of doubled haploid plants by androgenesis of cucumber (Cucumis sativus L.)

Six experiments (including pretreatment, embryonic callus induction media, preculture conditions, embryo induction media, embryo germination media, and genotypic effects) were conducted to develop an efficient cucumber (Cucumis sativus L., 2n = 2x = 14) anther culture protocol. Pretreatment and embryo induction were key factors for successful anther culture. Suitable temperature stress depended on the ecotype, i.e., cucumbers from cold areas responded well to cold shock whereas those from temperate areas responded well to heat treatment. The best medium for embryonic callus induction was MS medium supplemented with 4.44 μM BA, 2.26 μM 2, 4-D, 4.64 μM KIN, 3% sucrose and 0.8% agar. For embryo induction, MS medium supplemented with 0.54 μM NAA, 13.32 μM BA, 3% sucrose and 0.8% agar was optimal, and for embryo germination MS medium containing 2.22 μM BA, 6% sucrose and 1.2% agar was best. Using this protocol, we produced callus from 16 genotypes and regenerated plants from three of 20 evaluated. Three embryos per anther and 42 DH per 45 anthers (93% success) were obtained for cv. Ningjia No. 1, which was an improved result over a previous report. The origin of regenerants from microspores was determined by cytological, morphological and AFLP analyses.