Production of Embryogenic Callus and Plant Regeneration from Elite Guizhou Waxy Maize Inbred Lines

Immature embryos from three elite Guizhou waxy maize inbred lines （W21019, B7, and QCL5036） were evaluated for their ability of forming callus and regeneration into plants. Immature embryos harvested at different physiological stages were used as explants to initiate callus on N6 basal medium with 0-3.5 mg L-1 of 2,4-dichlorophenoxy acetic acid （2,4-D）. The concentration of 2,4-D, physiological age of immature embryos and genotype had a significant effect （P0.05） on the percentage of embryogenic callus formed. The optimum 2,4-D concentration for the initiation of embryogenic callus was varied among the waxy maize genotypes from 2.0 mg L-1 （B7 and QCL5036） to 3.0 mg L-1 （W21019）. The shoots were generated from embryogenic callus which were transferred into the regeneration medium supplemented with 0-2.5 mg L-1 of 6-benzylaminopurine （6-BA）. 6-BA in the medium significantly promoted the regeneration of embryogenic callus. Embryogenic size was also an important factor that affected regeneration capacity. 0.6-0.7 cm was proved to be the best size for regeneration from embryogenic callus and the mean number of shoots per primary callus in all genotypes achieved the highest number. The ability of the plant regeneration was also affected by genotype. W21019 had the highest number of shoots formed per primary embryogenic callus. With the optimum condition, the highest mean number of shoots per primary callus was up to 12.13, 5.73, and 3.33 in line W21019, B7, and QCL5036, respectively. The successful regeneration of the two inbred lines provides a basis for development of genetic transformation to improve priority traits such as enhanced insects and drought tolerance.

Factors Affecting Embryogenic Callus Production and Plant Regeneration in Anther Culture of Bupleurum chinense

Objective To evaluate the influences of the genotypes,anther developmental stages,and cultural conditions on the efficiency of embryogenic callus induction and plant regeneration in the anthers culture of Bupleurum chinense.Methods The different effects such as four genotypes,plant growth regulators,and temperature condition were compared in the experiments.The histological study was performed with the process of the anther culture.Results The highest inducing rate of embryogenic calli were achieved for the genotypes Zhongcaiyihao(ZCYH),Z4,and Z5 at the early-to middle-uninucleate stages,except for genotype ZPM1 at the tetrad stage.Cold pretreatment increased the production of the embryogenic callus,in which 4-day cold pretreatment improved the production of embryogenic callus from 0% to 2.2% and 5.0% for genotypes ZPM1 and ZCYH,respectively.No embryogenic callus was induced in the medium containing less than 0.75 mg/L 2,4-dichlorophenoxyacetic acid(2,4-D) .The highest regeneration rate(34.6%) was obtained in 1/2 MS salts regeneration medium supplemented with 0.1 mg/L 6-benzylmaminopurine(BA) .The low concentration of BA was able to promote the embryogenic callus formation and subsequent plantlet regeneration via somatic embryogenesis.Chromosome counting of regenerated plantlets showed mostly diploid plant(2n = 12) with only one haploid plant(n = 6) .Because of the low rate of microspore embryo formation,we only tracked the process of embryogenesis from the connective tissue,instead of microspore by histological observations.Conclusion This study establishes an efficient system for embryogenic callus induction and plant regeneration system.This is the first report on the haploid plantlet through the anther culture of B.chinense.

Sugarcane Callus Culture and Cytological Characteristics

In order to obtain sugarcane embryogenic calli for transgenosis, the effects of different soaking time of explants, medium, culture conditions and sampling time on sugarcane callus culture were investigated. The results showed that soaking ex- plants with MS liquid medium for 20 min could significantly reduce the browning rate; MS＋3.0 mg/L 2.4-D was appropriate for induction and proliferation of embryo- genic calli; dark condition was suitable for the culture of sugarcane calli; calli differ- entiated from materials collected in August exhibited low browning rate, high callus induction rate and high embryogenic callus induction rate. Calli with different pheno- types were stained, prepared into slides and observed under a microscope to ana- lyze the cytological characteristics. The resultsshowed that milky-white granular em- bryogenic calli had closely arranged cells with large nucleus and exhibited strong differentiation capacity, which were appropriate receptor materials for genetic trans- formation of sugarcane.

Comparative transcriptome study provides insights into acquisition of embryogenic ability in upland cotton during somatic embryogenesis

Background： The conversion from non-embryogenic callus （NEC） to embryogenic callus （EC） is the key bottleneck step in regeneration of upland cotton （Gossypium hirsutum）, and hinders the transgenic breeding of upland cotton. To investigate molecular mechanisms underlying acquisition of embryogenic potential during this process, comparation analysis of transcriptome dynamics between two upland cotton cultivars with different somatic embryogenesis abilities was conducted. Results： Differentially expressed genes involved in the transformation from NEC to EC were detected in the two different cultivars. Principal component analysis based on DEGs showed that the NEC tissues of the two cultivars were highly heterogeneous, whereas the derived EC tissues were similar, which suggested the homogeneousness of EC between different lines. In the highly embryogenic cultivar CCRI 24, more of these genes were down-regulated, whereas, in the recalcitrant cultivar CCRI 12, more were up-regulated. Bioinformatics analysis on these DEGs showed that the vast majority of differentially expressed genes were enriched in metabolism and secondary metabolites biosynthesis pathways. Flavonoid biosynthesis and phenylpropanoid biosynthesis pathways were enriched in both cultivars, and the associated genes were down-regulated more in CCRI 24 than in CCRI 12. We deduced that vigorous secondary metabolism in CCRI 12 may hinder primary metabolism, resulting in tardiness of cell differentiation. Interestingly, genes involved in the plant hormone signal transduction pathway were enriched in the recalcitrant cultivar CCRI 12, but not in CCRI 24, suggesting more radical regulation of hormone signal transduction in the recalcitrant cultivar. Signal transduction rather than biosynthesis of plant hormones is more likely to be the determining factor triggering NEC to EC transition in recalcitrant cotton lines. Transcription factor encoding genes showed differential regulation between two cultivars. Conclusions： Our study provides valuable information about the molecular mechanism of conversion from NEC to EC in cotton and allows for identification of novel genes involved. By comparing transcriptome changes in transformation from NEC to EC between the two cultivars, we identified 46 transcripts that may contribute to initiating embryogenic shift.

Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis

The induction and proliferation of embryogenic callus are key steps for large-scale propagation of somatic embryogenesis pathway and long-term preservation of coniferous germplasm.Callus can be induced from immature embryos of Korean pine(Pinus koraiensis Sieb.et Zucc.;Pinaceae)as explants,but there are problems,such as low proliferation efficiency,loss of embryogenicity,poor vigor;thus,best conditions for proliferation and culture of immature embryos of Korean pine are not yet clear.To solve the problems with somatic embryogenesis of Korean pine and determine the best culture conditions for callus induction and proliferation,we varied hormone concentration,subculture cycle of proliferation and other plant growth regulators combinations in media to induce callus formation by megagametophytes of three Korean pine families at different developmental stages,then analyzed the effects on embryogenic callus retention and cell proliferation using a quadratic regression orthogonal rotation design.The results showed that the family origin and collection date of explants significantly affected callus induction(induction rate reached 1.67%).Embryogenic maintenance and callus proliferation were best on DCR medium supplemented with 0.25 mg L^(−1)6-benzyl adenine,1 mg L^(−1)naphthaleneacetic acid,30 g L^(−1)sucrose,500 mg L^(−1),L-glutamine,500 mg L^(−1)casein hydrolysis and 6.5 g L^(−1)agar.In addition,the combination of 2,4-dichlorophenoxyacetic acid+6-benzyl adenine also had a better proliferative effect on callus.The effects of different combinations of growth regulators on callus proliferation efficiency were significantly different.Transfer to new medium every 13–15 days not only maintained robust callus vigor,but also yielded a larger proliferation coefficient.The techniques and conditions for embryogenic callus induction and proliferation of Korean determined here will serve as a foundation for establishing a large-scale system for somatic embryogenesis and propagation of Korean pine.

Effects of Auxins and Media on Callus Induction of Chinese Spring Wheat(Triticum aestivum L.)

The effects of auxins and media on callus induction from the mature and immature embryos of Chinese spring wheat （Triticum aestivum L.） varieties were investigated. It was found that genotype, medium, auxin source and concentration had the significant effects on the induction of embryogenic callus, explants germination and the increment of callus fresh weight. For immature embryos cultured on MS medium, 2 mg L^-1 of 2, 4-D was optimal, and the highest frequency of embryogenic callus （33.50%） was observed. For the mature embryos on N6 medium, 4 mg L^-1 of 2, 4-D was optimal. The frequency of embryogenic callus and increment of callus fresh weight on 2, 4, 5-T media were higher than those on 2, 4-D media, and in the presence of 2, 4, 5-T the precocious germination of explants for all genotypes were significantly suppressed. These results indicated that 2, 4, 5-T was superior to 2, 4-D and NAA in the culture of immature embryos. This is the first report about the effect of 2, 4, 5-T and NAA on wheat tissue culture, particularly in comparison with 2, 4-D in detail.

Induction and Genetic Identification of Embryogenic Calli from Hybrids of Shatian Pummelo

Shatian pummelo （Citrus grandis L. Osbeck cv. Shatian） is an elite variety in China, and the regeneration of the embryogenic callus is difficult. Diploid Shatian pummelo was used as the female and crossed with the allotetraploid somatic hybrid NS （Nova Tangelo ＋ Succari Sweet orange）, [ （ C reticulata Blanco x C. paradisi Macf.） cv. Nova ＋ C sinensis L. Osbeck cv. Succari]. About 90 days after pollination, the embryos obtained from crosses were cultured on the solid media of MT ＋ ME （malt extraction, 500 mg L^-1） and MT ＋ GA3 （1 mg L^-1）. The embryogenic callus was initiated from the embryoids and plantlets＇ hypocotyls and could be subcultured. Flow cytometry and SSR analysis verified that the callus was from the triploid hybrids. The callus had embryogenesis capacity and produced a large number of embryoids on MT ＋Lactose （50 g L^-1） medium after being subcultured for two years. It is comparatively easier to obtain the callus from the hybrid embryo than from Shatian pummelo itself. The callus is valuable for the conservation and utilization of Shatian pummelo.

Effect of Abscisic Acid on NaCI Stressed Callus Proliferation and Plant Regeneration in Rice

In rice, 2,4-Dichlorphenoxyacetic acid （MS2） has been considered best for callus induction and combination ofNAA, IAA, ABA （MS3） for somatic embryogenesis. Efficient plant regeneration was observed when MS basal salts supplied with 3% sorbitol and 3% maltose as carbon sources without hormones （MSac）. During this experiment, effect of sodium chloride （NaCl） and abscisic acid （ABA） was assessed on callus growth, plant regeneration and root induction efficiency of rice （Oryza sativa L.） varieties IR-6 and Basmati-370. Callus proliferation rate was highly decreased in both varieties on MS2b （100 mol.m3 NaCl ＋ 0.5 mg＇L1 ABA） than MS2a （100 mol＇m3 NaCl） cultures significantly. Proline, glycine-betaine and reducing sugars were increased significantly in MS2a and MS2b callusing cultures. However, total proteins were decreased in MS2a, while slightly increased in MS2b. Maximum plant regeneration （9.42 ±0.54 and 10.67 ±0.50 plantlets.callus1） from somatic embryos was observed on MS4c in IR-6 and Basmati-370, while 1.56 ± 0.06 （IR-6） and 0.95 ±0.05 （Basmati-370） plantlets callus1 were observed on MSab （100 mol·m3 NaCl）. No plant regeneration was observed on MS4b （100 mol·m3 NaCl ＋ 0.5 mg·L-1 ABA） medium in both varieties. Inhibition of root induction efficiency was high in MSsb （100 mol·m3 NaCl ＋ 0.5 mg·L-1 ABA） than MS5a （100 mol·m3 NaCl） in the stressed cultures （P 〈 0.05）. In this experiment, it was concluded that ABA involved in somatic embryogenesis and elevation of NaCl stress, while it causes inhibition of cell＇s growth as well as its regeneration into plantlets from somatic embryos.

Screening and verification of the factors influencing somatic embryo maturation of Larix olgensis

With embryogenic callus of Larix olgensisis, we investigated the effects of inositol, glutamine, casein hydrolysate, carbohydrate, abscisic acid and silver nitrate concentration on the maturation of the somatic embryo.Three dominant factors emerged, and we developed a response surface model based on the Box-Behnken design.We defined the optimal conditions for the maturation of somatic embryos. The contents of abscisic acid, silver nitrate, sucrose and casein hydrolysis significantly affected the amount of maturing embryos, but inositol, maltose and glutamine had no effect. By establishing a response surface model with multiple factors, we predicted that the optimal number of L. olgensis somatic embryos was 204 ± 4 gon basal medium, containing 18.28 mg Labscisic acid,5.46 mg Lsilver nitrate and 82.67 g Lsucrose. In the verification experiments, the addition of 20 mg Labscisic acid, 5 mg Lsilver nitrate and 80 g Lsucrose to BM yielded an average of 202.06 somatic embryos per gram. These results should guide large-scale breeding of L. olgensis.

Establishment and Optimization of the Regeneration System of Mature Embryos of Maize (Zea mays L.)

A reliable system was developed for regeneration from mature embryos derived from callus of four maize inbred lines （Liao 7980, Dan 9818, Dan 340, and Dan 5026）. The protocol was mainly based on a series of experiments involving the composition of culture medium. We found that 9 pM 2,4-dichlorophenoxyacetic acid in MS medium was optimum for the induction of callus. The induction frequency of primary calli was over 85% for four inbred lines tested. The addition of L- proline （12 mM） in subculture medium significantly promoted the formation of embryogenic callus but it did not significantly enhance growth rate of callus. Efficient shoot regeneration was obtained on regeneration medium containing 2.22 μM 6- benzylaminopurine in combinations with 4.64 μM Kinetin. Regenerated shoots were rooted on half-strength MS medium containing 2.85 μM indole-3-butyric acid. This plant regeneration system provides a foundation for genetic transformation of maize.

Optimization of the uidA Gene Transfer of Rosa hybrida via Agrobacterium tumefaciens: an Assessment of Factors Influencing the Efficiency of Gene Transfer

To develop a transformation protocol of Rosa hybrida Samantha via Agrobacterium tumefaciens, the authors examined the effect of different factors on T-DNA transfer by measuring transient expression levels of an intron-containing -glucuronidase gene. The results indicate that explant, light condition, salt concentration and acetosyringone (AS) concentration in co-culture medium are the most important factors, and factors like co-culture temperature, co-culture period and bacteria density have a strong effect on the growth of bacteria and then T-DNA transfer. Optimized co-cultivation was performed by inoculation of embryogenic callus with bacteria at a density of OD600= 0.50.8 for 20 min and co-culture in darkness under 23 C on medium with 1/2 MS salts and 300 mol稬1 AS for 3 d.

Somatic Embryogenesis in Lily Bulb Scale Cultures

Abstract： Somatic embryogenesis from lily bulb scales has not been studied in details, although tissue culture methods have been applied to the propagation for decades. The effects of different kinds and concentration of auxins for oriental lily somatic embryogenesis were investigated （Lilium hybrida var. Sorbonne）. 2, 4-dichlorophenoxyacetic acid （2, 4-D）, thidiazuron （TDZ） and α-naphthaleneacetic acid （NAA） media with benzyladenine（6-BA） and lactalbumin hydrolysate （LH） were used for embryogenic callus in the darkness. The best response on embryogenic callus formation was obtained on MS media supplemented 2, 4-D 2.0 mg·L^-1, 6-BA 0.5 mg·L^-1 and LH 300 mg·L^-1. Transfer embryogenic callus to the media with TDZ, 6-BA, kinetin （KT） supplemented 2, 4-D. The highest number of somatic embryos has been produced on medium with 0.5 mg·L^-1 2, 4-D and 0.3 mg·L^-1 KT. Germinated embryos with shoot axes were changed to MS media with 6-BA 0.5 mg·L^-1. The results suggest that in vitro culture of somatic embryogenesis from lily bulb scales can be used for plant regeneration.

Somatic embryogenesis and plant regeneration in Betula platyphalla

Betula platyphylla is a native tree species in northern China that has high economic and medicinal value.We developed an efficient protocol for the induction of somatic embryogenesis in B.platyphalla from immature zygotic embryos and assessed the effects of explant type,genotype,and plant growth regulators(PGRs)on embryogenic callus induction.Among the various explants evaluated,embryogenic callus was only produced from mature and immature zygotic embryos on medium with added 2,4-dichlorophenoxyacetic acid(2,4-D).Supplementation of 2,4-D-containing medium with cytokinins increased the frequency of embryogenic callus induction.On the 20 days after pollination,immature zygotic embryos that had been collected in mid-May yielded embryogenic tissue at the highest frequency(16.8%)when cultured on half-strength MS medium supplemented with 2.0 mg L^(-1)2,4-D and 0.2 mg L^(-1)6-benzylaminopurine(6-BA).The process of proliferation of embryogenic callus,somatic embryo formation,and subsequent plantlet conversion occurred under optimal culture conditions.When regenerated plants weretransplanted to soil,95%of them developed normally and grew vigorously.This somatic embryogenesis system required 3–4 months for the regeneration of B.platyphalla plantlets from immature zygotic embryos.

In vivo protective effect of late embryogenesis abundant protein(ApSK3 dehydrin)on Agapanthus praecox to promote post-cryopreservation survival

Dehydrins(DHNs),as members of the late embryogenesis abundant protein family,play critical roles in the protection of seeds from dehydration and plant adaptation to multiple abiotic stresses.Vitrification is a basic method in plant cryopreservation and is characterized by forming a glassy state to prevent lethal ice crystals produced during cryogenic storage.In this study,ApSK3 type DHN was genetically transformed into embryogenic calluses(EC)of Agapanthus praecox by overexpression(OE)and RNA interference(RNAi)techniques to evaluate the in vivo protective effect of DHNs during cryopreservation.The cell viability showed a completely opposite trend in OE and RNAi cell lines,the cell relative death ratio was decreased by 20.0%in ApSK3-OE EC and significantly increased by 66.15%in ApSK3-RNAi cells after cryopreservation.Overexpression of ApSK3 increased the content of non-enzymatic antioxidants(AsA and GSH)and up-regulated the expression of CAT,SOD,POD,and GPX genes,while ApSK3-RNAi cells decreased antioxidant enzyme activities and FeSOD,POD,and APX genes expression during cryopreservation.These findings suggest that ApSK3 affects ROS metabolism through chelating metal ions(Cu^(2+)and Fe^(3+)),alleviates H_(2)O_(2)and OH·excessive generation,activates the antioxidant system,and improves cellular REDOX balance and membrane lipid peroxidation damage of plant cells during cryopreservation.DHNs can effectively improve cell stress tolerance and have great potential for in vivo or in vitro applications in plant cryopreservation.

Transcriptome Profiling of Barley Cultivar Hua 30 MDEC in Response to Agrobacterium Infection

Agrobacterium-mediated transformation has been widely used in plants.However,the mechanism in plant cells’response to Agrobacterium infection was very complex.The mechanism of the determinants in host cell remains obscure,especially in barley,which is recalcitrant for Agrobacterium-mediated transformation.In the present study,microspore-derived embryogenic calli(MDEC)from barley elite cultivar were employed as unique subjects to characterize the mechanisms during the Agrobacterium infection process.Hua 30 MDEC can be successfully infected by Agrobacterium.RNA-sequencing at different infection points(0,2,6,12,24 hpi)was performed.The average expressional intensity of the whole genomics increased from 0 to 2 hpi,and then decreased subsequently.More upregulated than downregulated differentially expressed genes(DEGs)were counted at the same time.GO enrichment analysis showed that protein modification was significantly overrepresented in upregulated DEGs.Chromosome-related biological processes,gene expression and cellular metabolic processes were significantly overrepresented in downregulated DEGs.KEGG analysis showed that plant defense responses,phenylpropanoid biosynthesis and biosynthesis of amino acids were significantly enriched across the infection time course.Nine DEGs related to defense responses were identified.All DEGs were upregulated from 2 to 24 hpi.We speculate that these genes are possibly related to Agrobacterium infection.These findings will provide deep insights into the molecular events occurring during the process of Agrobacterium-mediated transformation.

Study on Cell Suspension Culture of Floribunda Rose

Friable callus was induced when immature seeds of floribunda rose were inoculated on MS medium supplemented with 2,4-D 3.0 mg.L^-1. When transfered onto subculture media, friable callus developed into embryogenic callus, which was used to establish cell suspension lines. Cell suspensions had to be subcultured at a interval of 4-5 days at the first several culture cycles. The best subculturing cycle for the stable cell suspensions was 8-10 days. The best inoculum quantity was 1 mL PCV（Packed Cell Volume） per 40 mL culture fluid.

Preliminary Investigation on Somatic Embryogenesis from Immature Cotyledon Explants of Shea （Vitellaria paradoxa G,）

Long juvenile phase and lack of effective protocols for large scale vegetative propagation are limitations to domestication and improvement of the shea tree. The present study seeks to develop a protocol for plant regeneration of shea （Vitellaria paradoxa） from immature cotyledon explants. Embryogenic callus cultures were induced on Murashige and Skoog medium （MS） containing 3% sucrose, 0.24% Phytagel, and various concentrations of 2,4-Dichlorophenoxyacetic acid （2,4-D） after four weeks of culture in darkness. Rates of embryogenic callus induction were significantly affected by the addition of 2, 4-D to the medium. Within 28 days of culture, the highest percentage of embyogenic calli （77.61%） occurred on MS media containing 0.45 ~tM of 2,4-D in the dark. Somatic embryos were obtained by culturing embryogenic callus （in the dark） on MS medium fortified with 3% sucrose, 0.24% phytagel and devoid of growth regulators. Culturing at 16 h photoperiod restricted both the induction of embryogenic calli cultures and somatic embryos. Somatic embryos germinated, developed shoots and rooted vigorously on MS medium devoid of growth regulators. Germinated plantlets were acclimatized, successfully.

Histological and Ultrastructural Observation Reveals Significant Cellular Differences between Agrobacterium Transformed Embryogenic and Non-embryogenic Calli of Cotton

Over the past few decades genetic engineering has been applied to improve cotton breeding. Agrobacterium medicated transformation is nowadays widely used as an efficient approach to introduce exogenous genes into cotton for genetically modified organisms. However, it still needs to be improved for better transformation efficiency and higher embryogenic callus induction ratios. To research further the difference of mechanisms for morphogenesis between embryogenic callus and non-embryogenic callus, we carried out a systematical study on the histological and cellular ultrastructure of Agrobacterium transformed calli. Results showed that the embryogenic callus developed nodule-like structures, which were formed by small, tightly packed, hemispherical cells. The surface of some embryogenic callus was covered with a fibrilar-like structure named extracellular matrix. The cells of embryogenic calli had similar morphological characteristics. Organelles of embryogenic callus cells were located near the nucleus, and chloroplasts degraded to proplastid-like structures with some starch grains. In contrast, the non-embryogenic calli were covered by oval or sphere cells or small clusters of cells. It was observed that cells had vacuolation of cytoplasm and plastids with a well organized endomembrane system. This study aims to understand the mechanisms of embryogenic callus morphogenesis and to improve the efficiency of cotton transformation in future.