Induction of Somatic Embryos in Arabidopsis Requires Local YUCCA Expression Mediated by the Down-Regulation of Ethylene Biosynthesis

Somatic embryogenesis is an important experimental model for studying cellular and molecular mechanisms of early embryo development. Although it has long been known that removal of exogenous auxin from medium results in somatic embryogenesis, the mechanisms underlying the initiation of somatic embryos （SEs） are poorly understood. In this study, we showed that YUCCAs （YUCs） encoding key enzymes in auxin biosynthesis are required for SE induction in Arabidopsis. To identify other factors mediating SE initiation, we performed transcriptional profiling and gene expres- sion analysis. The results showed that genes involved in ethylene biosynthesis and its responses were down-regulated during SE initiation. Ethylene level decreased progressively during SE initiation, whereas treatment with the metabolic precursor of ethylene, 1-aminocyclopropane-l-carboxylic acid （ACC）, or mutation of ETHYLENE-OVERPRODUCTION1 （ET01） disrupted SE induction, suggesting that ethylene plays a role in this process. Suppression of SE induction was also observed in the constitutive triple response 1 （ctrl） mutant, in which ethylene signaling was enhanced. These results indicate that down-regulation of not only ethylene biosynthesis, but also ethylene response is critical for SE induction. We further showed that ethylene disturbed SE initiation through inhibiting YUC expression that might be involved in local auxin biosynthesis and subsequent auxin distribution. Our results provide new information on the mechanisms of hormone-regulated SE initiation.

Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L.‘Moldova’

We examined the somatic embryogenesis from and histological studies of zygotic embryos of seeds in European Grape ＇Moldova＇ （Vitis vinifera U ＇Moldova＇）. Primary calli were initiated on Nitsch and Nitsch （NN） medium supplemented with 1.0 mg·L^-1 2,4-D and 0.5 mg·L^-1 6-BA. Embryogenic calli were produced upon transfer to a NN medium with 0.5 mg·L^-1 6-BA and 2 mg·L^-1 NAA and somatic embryos were obtained on a half strength MS medium without plant growth regulators. During the somatic embryo germination, an addition of 1.0 mg·L^-1 6-BA in the medium could accelerate somatic embryos to develop into normal plants and increase the conversion rate from 0 to 43.3%. Histological studies of embryogenic calli and somatic embryos demonstrated dynamic changes of proteins and starch grains. The developmental processes of somatic embryos were similar to those of zygotic embryos, including typical epiderma, cotyledon primordium and vascular tissue.

Effect of plant growth regulators on direct somatic embryogenesis in camphor tree(Cinnamomum camphora L.)from immature zygotic embryos and embryogenic calli induction

A description of a successful direct somatic embryogenesis induction from immature zygotic embryos of a camphor tree （Cinnamomum camphora L.） is presented. After a subculture of 2-3 years, embryogenic calli could be derived from primary somatic embryos. Immature zygotic embryos were cultured on a Murashige and Skoog （MS） basal medium supplemented with a range of combinations of cytokinins （BA） and auxins （2,4-D or NAA） for somatic embryo induction. Primary somatic embryos could be induced directly in almost all PGR combinations. A positive effect of 2,4-D on somatic embryogenesis from immature zygotic embryos of camphor tree was obtained. BA at appropriate concentrations （〈 5 mg-L-1） had an effect similar to 2,4-D, whereas high concentrations （〉 5 mg·L^-1） of BA had the effect of restraining somatic embryo induction. NAA had a less positive effect on somatic embryogenesis than 2,4-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.

Expression analysis of OsSERK,OsLEC1 and OsWOX4 genes in rice(Oryza sativa L.)callus during somatic embryo development

Somatic embryogenesis is an asexual reproduction process that occurs in many plant species,including rice.This process contains several totipotency markers such as Somatic Embryogenesis Receptor-like Kinase(SERK),Leafy Cotyledon1(LEC1)and WUSCHEL-Related Homeobox4(WOX4)and also a helpful model for embryo development and clones and transformations.Here,we report the gene expression during somatic embryo development correlates with regeneration frequency in 14 Javanica rice(pigmented and non-pigmented)using modified N6 media supplemented with Kinetin(2.0 mg/L)and NAA(1.0 mg/L).Although there have been advances in understanding the genetic basis of somatic embryogenesis in other varieties,rice is still unexplored,especially during somatic embryo development.Moreover,for the formation of callus induction from immature embryos,2,4-D(2.0 mg/L,3.0 mg/L)was used.This study analysed the gene expression of OsSERK,OsWOX4 and OsLEC1 genes through RT-PCR analysis.Higher expression of the OsLEC1 gene indicates that their function may correlate in the in vitro with the high response of rice after transfer to regeneration media.This study found that rice varieties of pigmented rice(MS Pendek and Gogoniti II)and non-pigmented rice(Pandan Ungu)showed high regeneration frequency,showing higher OsLEC1 expression than other varieties because OsLEC1 promotes the maturation of somatic embryos in plant regeneration on day 14.However,the contrast with Genjah nganjuk may be effective because of other regulatory genes.RT-PCR analysis showed OsSERK had less expression level than OsLEC1 and OsWOX4 in the varieties,which correlate with the percentage of plant regeneration,but not for Gogoniti II.In conclusion,the higher percentage of plant regeneration correlates with the higher expression level of OsLEC1 at day 14 of media regeneration of rice.

Morphology of somatic embryogenesis and plantlet formation in tissue cultures of lantern tree (Koelreuteria bipinnata var.integrifoliola)

Somatic embryogenesis ofKoelreuteria bipinnata var. integrifoliola was observed, plantlet formation in different types of somatic embryos was studied and the effect of abnormal embryos on plantlet formation was identified. Results show that somatic embryos of K. bipinnata var. integrifoliola include normal embryos, embryos with abnormal cotyledons, vitrified embryos, albino embryos, secondary embryos, linked embryos, embryos with abnormal growing points and embryos with expanding hypocotyl. After 40 d of callus culture, the response of normal somatic embryos from K. bipinnata var. integrifoliola was 26.7%, embryos with abnormal cotyledon 30.3% while other types of somatic embryos were below the 20% level. Most of normal embryos developed into plantlets and plantlet formation reached 94.9%. But the percentage of plantlet formation decreased apparently in abnormal embryos： the number of embryos with abnormal cotyledon declined to 76.1%, that of linked embryos to 47.4% and other types of abnormal embryos to below the 20% level. Albino embryos and embryos with abnormal growing point did not develop at all into plantlets. Embryos with abnormal cotyledons, linked embryos and embryos with abnormal growing points were observed during early stages of somatic embryogenesis, but vitrified, secondary and albino embryos and calli of embryos were observed at later stages. Increasing sucrose concentrations can decrease the occurrence of vitrified embryos, but the number of albino embryos decreased with an in- creasing in sucrose concentration.

Plantlets Regeneration via Somatic Embryogenesis from the Nucellus Tissues of Kinnow Mandarin (Citrus reticulata L.)

Studies were initiated to explore the role of nucellus tissues and growth regulators in plantlets regeneration via somatic embryogenesis of Kinnow mandarin [Citrus reticulata L. (Blanco)]. Nucellus tissues were cultured on MS medium supplemented with different concentrations and combinations of auxins, cytokinins and malt extract for primary callus induction. The best response for primary callus induction (90%) was obtained when MS medium was supplemented with 5 mg/l 2,4-D and 500 mg/l malt extract. Best results for embryogenic callus induction (80%) were obtained in C8 medium. The induction of somatic embryos was highest when MS medium was supplemented with 1 mg/l BAP and maturation of somatic embryos occurred when MS medium was supplemented with 5 mg/l 2,4-D and 1 mg/l BAP. Maximum plantlets were regenerated (92%) from the somatic embryos on half strength MS medium with no hormones. The plantlets were successfully acclimatized in different potting mixtures and highest survival rate (100%) was achieved in potting mixture containing sand and peat moss (2:1).

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.

Effect of Rare Earth Elements on the Induction Frequency of the Somatic Cell Embryo in The Fruit of Chinese Wolfoerry

A part of lanthanides could raise the induction fraquency(IF) of the somatic cell embryo(SCE) in the fruit of Chinese wolfbeny. The effect of thorium and yttrium used for this purpose is not obvious and even plays a role of inhibition when they are used with a concentration of more than 4 ppm.When the combinations of different rare earth elements (RE) are used, the diversity of the effects amongthem is large. Some of them help to raise the iF of the SCE while the others inhibit the generation of SCE.The mischmetal results in the best effect, giving a relative IF of 295.4%.

Breeding and Simple-rapid Regeneration Protocol for Jisheng 1 with Glandless Trait and High-frequency Somatic Embryo Production Ability

Most of model cotton varieties used in tissue culture have glands on both the reproductive and vegetative parts of the plant.These glands contain compounds that are toxic to human and non-ruminant animals.The presence of these compounds limits their usage as food and feed.To obtain a glandless cotton variety with high-frequency somatic embryo production ability,27 glandless varieties

Evaluation of somatic embryo production during embryogenic tissue proliferation stage using morphology,maternal genotype,proliferation rate and tissue age of Pinus thunbergii Parl

To determine the optimal embryogenic capacity(somatic embryo production)of the selected elite nematode-resistant genotypes of Pinus thunbergii,variables such as embryogenic tissue(ET)morphology,maternal genotype,proliferation rate and tissue age were analyzed.ET morphology and histological evaluation of the proliferation stage showed a decrease in filamentous clump and protuberant surfaces and a decline in the acetocarmine-staining area,which indicates a decrease in somatic embryo production(SEP).Variations in cell physiology during the prolifera-tion stage showed that SEP was positively correlated with soluble sugars and proteins,but negatively correlated with starch,peroxidase,and superoxidase.In addition,SEP was significantly(p<0.001)affected by maternal genotype,tis-sue age and proliferation rate.Moreover,SEP was positively correlated with proliferation rate(r=0.98,p<0.001),but negatively correlated with tissue age(r=−0.95,p<0.001).In general,the results suggest that SEP could be assessed in ET proliferation stages by the apparent cell morphology,histology,proliferation rate and tissue age,which provides novel insights for evaluating the ET maturation capacity(number of somatic embryos)during the proliferation stage of P.thunbergii somatic embryogenesis.

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.

Specific Proteins in the Indirect Somatic Embryogenesis of Freesia Refracta

Using the young inflorescence segments of Freesia refracta as explants, indirect somatic embryogenesis of somatic cells was induced in a N6 medium supplemented with some exogenous hormones. SDS-polyacrylamide gel electrophoresis（SDS-PAGE） was used for the analysis of soluble proteins produced during the somatic embryogenesis of this plant. There are six polypeptides, which might play some roles in the process of somatic embryo development. Tltree polypeptides（45, 53 and 55 kD） were detected in the stages of embryogenic callus, globular embryoid, and embryoid with coleoptiles, except the embryoid with leaf. One polypeptide（ 83 kD） was specific for the stages of embryoid with eoleoptiles and embryoid with leaf. One polypeptide（37 kD） was detected in the first two stages, namely, embryogenic callus and globular embryoid. One polypeptide（35 kD） was regularly synthesized in each stage, from embryogenic callus to embryoid with leaf.

Agrobacterium tumefaciens-mediated transformation of modern rose(Rosa hybrida)using leaf-derived embryogenic callus

Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.

Synthetic Seed Preparation, Germination and Plantlet Regeneration of Litchi (Litchi chinensis Sonn.)

Litchi chinensis sonn.) ranks second after mango amongst the most important fruit crops cultivated worldwide. Litchi is a very valuable crop throughout the world because it is a table fruit and wines are also produced from it. The existing cultivars are highly polyploidy and heterozygous in nature. It is propagated through air layering and marcottage methods and storability is very low. Synthetic seeds can be stored for a long time and its genetic constitution could remain the same. For germplasm maintenance and clonal propagation, synthetic seeds can be used. Somatic embryogenesis has been reported from anther or embryogenic suspension culture in various species of litchi. Regeneration via organogenesis and somatic embryogenesis from zygotic embryos has also been reported in certain species. Developing a methodology for getting somatic embryogenesis with a high frequency from zygotic embryos which is available once in a year, would be particularly useful for genetic improvement of litchi. Cotyledonary stage somatic embryos developed from zygotic embryos were encapsulated in 2% alginate gel. The encapsulated somatic embryos (ESEs) germinated successfully on 0.7% agar medium containing 3% sucrose concentration in NN basal medium (half strength of major and minor salts) with 1 mg·l-1 of gibbrellic acid. Percentage germination and plantlet development for ESEs was higher than that of non encapsulated embryos (NSEs). In comparison to different hormones, gibberellic acid has a significant influence on the germination rate of ESEs after one week of dehydration was seen maximum at 9% sucrose and abscisic acid (1 mg·l-1) in half strength of major and minor salts in Nitsch and Nitsch medium resulting in extended storage up to 90 days without loss in germination potential and capability to regenerate into plantlets. Normally developed plantlets regenerated from ESEs were successfully adapted to soil to obtain a full grown plant.

Precocious In-Vitro Flowering of Perennial Asparagus (Asparagus officinalis L.) Regenerants with a Chemical Inducer

A precocious flowering system of regenerants in asparagus (Asparagus officinalis) was achieved by treatment with a chemical inducer. Somatic embryos withered completely by being processed for 8 - 12 days with 200 μM n-propyl N-(3,4-dichloro-phenyl)carbamate that had been dissolved in distilled water. In contrast, precocious flowering occurred at an extremely low rate (3.4%) when somatic embryos were processed in carbamate dissolved in Murashige and Skoog’s liquid medium. To encapsulate the female and male embryos, we surveyed the optimum conditions of viscosity and concentration of sodium alginate for encapsulating the seeds, and we screened the values of 80 - 120 cps and 2% - 3%, respectively. The synthetic seeds produced also withered when they were processed with the carbamate dissolved in distilled water. However, when Murashige and Skoog’s liquid medium was used for the solvent, the flowering frequency of the synthetic seeds was enhanced (13.3%). Based on our morphological and histological observations, female and male regenerants that were processed with the carbamate solution produced individual flower organs. The conversion of sex expression did not occur. A precocious flowering system would allow a significant reduction in the time required for perennial seedlings to flower and can, therefore, save time required for further experiments that employ floral homeotic mutants.

Generation of ApoE deficient dogs via combination of embryo injection of CRISPR/Cas9 with somatic cell nuclear transfer

Atherosclerotic cardiovascular disease is the leading cause of death in the world which is resulted from complex interactions among multiple genetic and environmental factors （WHO）. Athero- sclerosis is a chronic inflammatory disease characterized by accumulation of lipids in the arterial wall （Gofman and Lindgren, 1950）. Tremendous clinical and experimental efforts have been made to reveal the pathogenesis of the disease. Nevertheless, the mechanism of atherosclerosis is still unclear. A suitable animal model to study metabolic disorders and subsequent atherosclerosis is a necessity. The traditional method by feeding high fat diet to establish animal models of atherosclerosis disease is time- consuming and laborious, and in many circumstances, the pheno- types are not consistent among the individual models.