Improvement of Plant Regeneration from Immature Embryos of Wheat Infected by Agrobacterium tumefaciens

Wheat, one of the most important food crops, has been extensively studied with respects to plant regeneration and transformation employing the immature embryos as recipient tissues. However, the transformed tissues often become severely necrotic after co-cultivation with Agrobacterium, which is one of the major obstacles in gene delivery. In this study, wheat varieties CB037, Kenong 199, Xinchun 9, Lunxuan 987, and Shi 4185 showed desirable culture potential or high infection ability in Agrobacterium-mediated transformation. Similarly, optimal regeneration conditions were determined by testing their ability to inhibit the cell necrosis and cell death phenotype. Two auxins of 2,4-dichlorophenoxyacetic acid （2,4-D） and 3,6-dichloro-o-anisic acid （dicamba） were evaluated for highly significant effect on both callus and plantlet production, although they were genotype-dependent in wheat. Substitution of 2,4-D by dicamba enhanced the growth and regeneration ability of callus from the immature embryos of most genotypes tested. The callus growth state couldn’t be modified by adding antioxidants such as ascorbic acid, cysteine, and silver nitrate or organic additives such as thiamine HCl and asparagine to the media. On the contrary, the best tissue statement and plant regeneration was achieved by employing the media containing the simplest MS （Murashige and Skoog） components and dicamba without organic components and vitamins. Thereby, our results are thought to inhibit wheat cell necrosis effectively and suggested to be used for more wheat genotypes.

Effects of Environmental Temperature on the Regeneration Frequency of the Immature Embryos of Wheat(Triticum aestivum L.)

The immature embryos （IEs） of wheat are the most widely used tissues for in vitro culture and genetic transformation due to its high regeneration competency. However, this explant can only be maintained in 4℃ daily cooler for a short period time for its use in plant tissue culture or transformation experiments. This study aimed to investigate the effects of environmental temperature, cryopreservation storage temperature, and heat shock culture （HSC） temperature on the regeneration frequency of wheat IEs. Results indicated that environmental temperature significantly affected the induction of embryonic calli. The optimum total accumulated temperature （TAT） during the time of anthesis and sampling for regeneration of these tissues was around 280℃ for spring wheat type cv. CB037 and approximately 300℃ for winter wheat type cv. Kenong 199. Regeneration ability obviously declined when the highest environmental temperature was over 35℃ for 1 d or a high temperature between 30 and 33℃ lasted for 5 d during anthesis and sampling. This finding was verified by culturing the freshly isolated IEs under different temperatures from 29 to 37℃ in different controlled growth incubators for 5 d; the IEs almost completely lost regeneration ability when the temperature rose to 37℃. Cryopreservation of-20℃ caused the wheat samples lost ability of producing callus or embryonic callus in a few days, and cryopreservation of-10℃ more than 10 d made the regeneration potential of the tissues dramatically declined. Comparatively, the temperature that best maintained high regeneration ability was -5℃, at which the materials can be maintained for around 1 mon. In addition, the preservation of the immature samples at -5 or -10℃ inhibited the direct germination of the IEs, avoiding the embryo axis removing process. Our results are useful for ensuring that field collection and cryopreservation of the wheat IEs are done correctly to enable tissue culture and genetic transformation.

Effects of inter-culture, arabinogalactan proteins, and hydrogen peroxide on the plant regeneration of wheat immature embryos

The regeneration rate of wheat immature embryo varies among genotypes, howbeit many elite agriculture wheat varieties have low regeneration rates. Optimization of tissue culture conditions and attempts of adding signal molecules are effective ways to increase plant regeneration rate. Inter-culture is one of ways that have not been investigated in plant tissue culture. Moreover, the use of arabinogalactan proteins （AGPs） and hydrogen peroxide （H202） have been reported to increase regeneration rate in a few plant species other than wheat. The current research pioneeringly uses inter-culture of immature embryos of different wheat genotypes, and also investigates impacts of AGP and H2O2 on the induction of embryogenic calli and plant regeneration. As a result, high-frequency regeneration wheat cultivars Kenong 199 （KN 199） and Xinchun 9 （XC9）, together with low-frequency regeneration wheat line Chinese Spring （CS）, presented striking increase in the induction of embryogenic calli and plant regeneration rate of CS through inter-culture strategy, up to 52.19 and 67.98%, respectively. Adding 50 to 200 mg L-1 AGP or 0.005 to 0.01‰ H2O2 to the callus induction medium, enhanced growth of embryogenic calli and plant regeneration rate in quite a few wheat genotypes. At 50 mg L-1 AGP application level in callus induction medium plant regeneration rates of 8.49,409.06 and 283.16% were achieved for Jimai 22 （JM22）, Jingdong 18 （JD18） and Yangmai 18 （YM18）, respectively; whereas at 100 mg L-1 AGP level, CS （105.44%）, Chuannong 16 （CN16） （80.60%） and Ningchun 4 （NC4） （62.87%） acted the best. Moreover CS （79.05%）, JM22 （7.55%）, CN16 （101.87%）, YM18 （365.56%）, Yangmai 20 （YM20） （10.48%）, and CB301 （187.40%） were more responsive to 0.005 %o of H2O2, and NC4 （35.37%） obtained the highest shoot regeneration rates at 0.01%o of H2O2. Overall, these two methods, inter-culture and AGP （or H2O2） application, can be further applied to wheat transgenic research.

Optimization of Callus Induction Conditions from Immature Embryos in Maize and Plant Regeneration

This research uses the immature embryos of inbred maize lines(GSH9901,Hi01,Hi02,and Chang 7-2)as receptor materials to establish the callus induction system.These inbred lines provide the receptor materials for the genetic regeneration of maize and the verification of the genetic functions of maize.The factor experiment and orthogonal experiments were used to investigate the impacts of different genotypes,immature embryo size,shield orientation,2,4-D concentration,proline concentration,and folic acid concentration on the induction rate of embryogenic callus tissue.A sensitivity experiment testing glyphosate(Bar)and an antibiotic(Cefotaxime sodium)were also conducted.The results indicate that the immature embryos of inbred maize line GSH9901 were the most effective for callus tissue induction,and the immature embryos with a length of 1.6-2.0 mm produce the best result.The upward shield face is more successful for the formation of induced callus.Using orthogonal analysis,we found that the optimal combination for the induction system was A_(3)(2,4-D concentration 0.25 mg mL^(-1)),B_(1)C_(3)(proline concentration 0.8 mg mL^(-1)),and D 2(folate Concentration 0.5 mg mL^(-1))and the induction rate reached 84%.We found that cold storage at 4℃ for 1 d is more conducive for the formation of embryogenic callus than the other treatments tested.The sensitivity experiment for callus tissue screening revealed the critical concentration of glyphosate to be 10 mg ml^(-1),and the critical concentration of antibiotic is 250 mg ml^(-1).Using this combination of glyphosate and antibiotic resulted in regenerated plants.This study established the optimal conditions for immature embryo callus tissue induction in maize.

Study on Callus Induction from Immature Embryos and Plant Regeneration of Different Genotypes of Maize

The induction, subculture and differentiation of callus from immature embryos of maize （ Zea mays L. ） inbred line were studied and optimized. The re- suits revealed that, 2 mg/L 2,4-D was the optimal concentration to induce embryonic callus. Calli induced from inbred Qi319 and LY92 had good morphology and high regeneration. The embryos of the two inbred lines were selected as explants to establish efficient and stable tissue culture and transformation system.

Optimization of Electroporation Parameters for Immature Embryos of indica Rice (Oryza sativa)

To obtain a suitable condition for electroporation transformation in indica rice, the 10-day-old immature embryos were selected for optimization experiments. The results showed that one pulse at 850 V/cm, 950μF capacitance, 200 μL electroporation buffer with 70 mmol/L sodium glutamate, 100 μg/mL plasmid, 50μg/mL carrier DNA, 20 embryos per cuvette, 0℃ treatment and CC medium were the best parameters, which not only improved the transformation efficiency to 30.89%, but also ameliorated the embryo survival ratio to 95.92%. To further verify the practicability of this condition, the embryos from another indica rice variety and a rice type Ⅱ metallothionein-like gene （OsMT2bL） promotec：mgfp5：：gusA construct were tested, and specific GUS expression on the embryos was visualized by histochemical staining. The results showed that the GUS expression on the embryos activated by the OsMT2bL promoter was mainly concentrated on the apical point of the plumule whereas the expression driven by CaMV35S promoter was distributed on nearly all areas of the electroporated tissues. These results indicated that the optimized embryo electroporation conditions could be used not only in genetic transformation of indica rice but also in assay of gene regulation on embryos.

In vitro culture of immature embryos from Koelreuteria bipinnata var. integrifoliola

For the mass production of Koelreuteria bipinnata var. integrifoliola with selected, hybrid or genetically engineered genotypes, one potentially desirable propagation strategy is based on embryo culture. The immature embryo development in vitro from K. bipinnata var. integrifoliola was studied under different conditions of embryo age, basic culture media and plant growth regulators. The results show that： 1） germination rate of grade 3 embryos in immature seeds with 0.6-0.8 cm diameter was 98.9%. The germination rate of grade 2 embryos in immature seeds with 0.4-0.6 cm diameter was 77,8% and the germination rate of grade 1 embryos in immature seeds with 0.4 cm diameter was 15.6%. 2） The amounts of macroelements in MS medium had no clear effect on the germination rate of immature grade 3 embryos and had a modest effect on plantlet growth, where the best medium was MS or 1/2 MS. The rates were all greater than 90%. 3） The germination rate of grade 3 embryos was greater than 87% when the medium contained a low concentration of NAA or no plant growth regulators at all and decreased markedly when BAP alone or BAP and NAA together were added to the media. We suggest that in vitro culture of immature embryos from K. bipinnata vat. integrifoliola can be enhanced when a small amount of plant growth regulators is added. The addition of BAP has an adverse reaction to the germination and development of immature embryos.

Transcript Profiling Reveals Abscisic Acid,Salicylic Acid and Jasmonic-Isoleucine Pathways Involved in High Regenerative Capacities of Immature Embryos Compared with Mature Seeds in japonica Rice

Induced pluripotent cell mass plays a role in genetic transformation mediated by Agrobacterium. Mature seeds are more recalcitrant to the induction of suitable calli than immature embryos in rice, but the exact molecular mechanisms involved remain elusive. In this study, the morphological structure of calli induced from mature seeds and immature embryos were observed under a scanning electron microscope using a paraffin embedded technique. Meanwhile, a total of 2 173 up- and down-regulated genes were identified in calli induced from mature seeds and immature embryos by RNA-seq technique and furtherly confirmed by quantitative real-time PCR. The results revealed the remarkable morphological differences in calli induced from mature seeds and immature embryos, and plant hormone signal transduction and hormone biosynthesis pathways, such as abscisic acid, salicylic acid and jasmonic-isoleucine, were found to play roles in somatic embryogenesis. This study provided comprehensive gene expression sets for mature seeds and immature embryos that were served as an important platform resource for further functional studies in plant embryogenesis.

Optimization of Callus Induction Conditions from Immature Embryos of Maize under Stress

The embryos of maize(Zea mays L.)inbred lines GS02,GS07,GS08,GS11 and GS15 were used as receptor materials to optimize the receptor system from the aspects of genotype,medium components and stress(PEG6000,mannitol,salt and low phosphorus).The results showed that GS07 had the highest induction rate(95.2%).Orthogonal test analysis showed that the best combination of medium components in induction was A2B3C1D3(2),namely,the concentration of 2,4-dichlorophenoxy acetic acid(2,4-D)was 4 mg·mL^(-1),the concentration of L-Proline(L-Pro)was 0.8 mg·mL^(-1),and the concentration of silver nitrate(AgNO3)was 10 mg·mL^(-1)(or 5 mg·mL).Interestingly,we found that the optimal medium supplemented with 30 g·L^(-1)PEG6000 or 80 g·L^(-1)mannitol was suitable for antioxidant enzyme activity and malondialdehyde(MDA)content in GS07 callus.Exogenous 10 mmol·L^(-1)Ca^(2+)in the medium components with 100 mmol·L^(-1)sodium chloride(NaCl)could improve the activity of antioxidant enzymes in GS07 callus.Callus of GS07 could divide normally and grow well in medium components with 27 mg·L^(-1)KH_(2)PO_(4).This study enhanced the adaptability of maize callus to stress and optimized the culture conditions.

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize(Zea mays L.)

Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.

Cytological Correlation Between Flower and Vegetative Shoot Development from Immature Embryo-Derived Calli in Wheat

In this paper, the traits of immature embryo culture from 45 genotypes of bread wheat were investigated in detail, and trait correlation analysis in combination with histomorphological examination was used to study the cytological origin of flower as well as the correlation between it and vegetative shoot development from the calli. The rate of floral development in the subculture was highly significantly and positively related to that of the differentiation culture, which reveals that the genetic system of floral development from immature embryo-derived calli in wheat was highly correspondent and stable. The rate of floral development was related neither to the traits in the dedifferentiation culture, nor to the vegetative shoot formation from the total induced calli, but significantly and positively related to that of the calli without leaf-like green spots in the differentiation culture. These findings indicate the partial correlation as well as differentiation between flower and vegetative shoot development. The floral development might only be associated with the genetic system controlling organ regeneration, while vegetative shoots formed from the two ways, including somatic embryogenesis and organ regeneration.

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 P1 and P2 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 results of the two methods, generation joint analysis may genetic information. to be different using the two methods. By comparison of the not only raise experimental precision but also provide more

A Preliminary Study on Immature Embryo Culture and Plant Regeneration of Lagerstroemia indica

[ Objective ] This study aimed to explore immature embryo culture of Lagerstroemia indica and investigate the appropriate conditions for growth and differentiation. [ Method] Immature embryos of L. indica were employed as the explants for germination induction to establish aseptic lines. Based on that, the effects of different hormone levels and culture conditions on immature embryo culture of L. indica were analyzed. [ Result ] Peeled immature embryos of L. indica were germinated easily, leading to a germination rate of 100%. The optimal initial medium was MS ＋ BA0.5 ＋ NAA0.1 ＋ sucrose 3.0% ＋ agar 0.7% ; the optimal shoot induction medium was MS ＋ BA0.5 ＋ NAA0.1 ＋ sucrose 3.0% ＋ agar 0.7% ＋ coconut milk 10% ; the optimal rooting medium was MS ＋ BA0.5 ＋ IBA0.1 ＋ sucrose 3.0% ＋ agar 0.7% ＋ coconut milk 10%. [ Conclusion] This study provided a technical reference for subsequent optimized breeding of L. indica.

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.

Constructing the barley model for genetic transformation in Triticeae

Barley（Hordeum vulgare L.） is one of the oldest domesticated crops, showing dramatic adaptation to various climate and environmental conditions. As a major cereal crop, barley ranks the 4th after wheat, maize and rice in terms of planting area and production all over the world. Due to its diploid nature, the cultivated barley is considered as an ideal model to study the polyploid wheat and other Triticeae species. Here, we reviewed the development, optimization, and application of transgenic approaches in barley. The most efficient and robust genetic transformation has been built on the Agrobacterium-mediated transfer in conjunction with the immature embryo-based regeneration. We then discussed future considerations of using more practical technologies in barley transformation, such as the T-DNA/transposon tagging and the genome editing. As a cereal crop amenable to genetic transformation, barley will serve as the most valuable carrier for global functional genomics in Triticeae and is becoming the most practical model for generating value-added products.

Optimization of Agrobacterium tumefaciens-Mediated Genetic Transformation of Maize

Immature embryos of inbred maize(Zea mays)lines(H8183,H8184,and H8185)were used for Agrobacterium infection.We used theβ-glucuronidase gene(GUS)as the target gene and the glufosinate resistance gene(bar)as the selection marker.We conducted research on several aspects,such as different genotypes,coculture conditions,screening agent concentrations,and concentrations of indole-3-butytric acid(IBA),6-benzylaminopurine(6-BA),and ascorbic acid(Vc)in the differentiation medium.We optimized the genetic transformation system,and the obtained results indicated that among the three lines studied,the induction rate of H8185 was the highest at 93.2%,followed by H8184,with H8183 having the lowest induction rate(80.1%).The best coculture method was that using the N6 coculture medium layered with a sterile filter paper.Using orthogonal analysis,we found that the optimal combination of the three factors in the differentiation medium was A_(3)(1 mg mL^(−1)IBA),B_(3)C_(1)(1.6 mg mL^(−1)6-BA),and D_(3)(1.5 mg mL^(−1)Vc).Through GUS staining analysis,Bar test-strip analysis,and polymerase chain reaction,five transgenic plants were finally obtained.This study established the optimal conditions for genetic transformation in maize.

The Selection of Transgenic Recipients from New Elite Wheat Cultivars and Study on Its Plant Regeneration System

In the protocol of wheat transformation, to use elite wheat cultivars as exogenous gene recipients can speed up the process of commercial field applications of transgenic wheat. However, it is necessary to screen wheat cultivars with good tissue culture response （TCR） continuously from plenty of elite wheat cultivars released for wheat transformation, and it is also important to find a plant regeneration system that is suitable for these cultivars. So, the TCR of mature and immature embryos of six wheat cultivars Chuannong 11 （CN11）, Chuannong12 （CN12）, Chuannong17 （CN17）, Chuannong18 （CN18）, Chuannong19 （CN19）, and Chuannong21 （CN21）, which possess superior agronomic traits, were investigated by using a good TCR wheat cultivar Bobwhite as control. The results indicated that only the immature and mature embryos of CN12, CN17, and CN18 exhibited good TCR compared with Bobwhite. No significant differences were observed between embryos of Bobwhite and of the three cultivars in TCR. Mature embryo-derived calli of CN12 were used as explants for transformation by particle bombardment of SAMDC gene. Seven transformants were obtained and the efficiency was 2.3%. This research supplies three new elite recipient cultivars for wheat transformation. The wheat plant regeneration system used in this research is different from those successful ones reported previously and it could be a reference for other wheat genotypes. Furthermore, Bobwhite and the three wheat cultivars were proved to be 1RS/1BL translocation, by methods of A-PAGE, C- banding, and genomic in situ hybridization （GISH）. These results imply that probably there is some relationship between 1RS/1BL translocation and TCR of wheat embryos. So this research gives us a hint that we should pay more attention to the 1RS/1BL translocations when we screen the wheat cultivars with good TCR and also that the mechanism of the effect of 1RS/ 1BL translocation on TCR is worthy of being investigated.

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.

The Application of Somaclonal Variation in Early Maturity,High Yield and High Quality Improvement in Wheat

Yield characters, maturity and grain protein content of somaclones derived both from immature embryo of cultivar 77(2)-Spring and single-cell culture of cultivar NE7742 in vitro were studied and the wide variation was found. Somaclones with maturity 8 days earlier than or the same as CK NE 7742 (high yield, early maturity and high quality), combining with high quality (grain protein content 15.5% - 18%) and high yield (the same as 7724 or higher) have been found and selected and now multiplied for 8 generations. The results of cultivar comparison trial in 1995 showed that several somaclones (the yields were significantly higher than CK DN120) could be used directly in wheat production. The studies confirmed that somaclonal variation is one of the effective ways for early maturity, high-yielding and high-quality improvement in wheat.