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.

Role of hydrogen peroxide in stress-induced programmed cell death during somatic embryogenesis in Fraxinus mandshurica

We examined how reactive oxygen species, in the form of hydrogen peroxide (H2O2), affect osmotic stress–induced programmed cell death during somatic embryogenesis from cotyledon explants of Manchurian ash (Fraxinus mandshurica Rupr.). We found that substantial osmotic stress was essential for Manchurian ash somatic cells to obtain embryogenic competence. The explant cells displayed hallmarks of programmed cell death, chromatin condensation, and DNA fragmentation to oligonucleotides during somatic embryogenesis. Increasing concentrations of plant growth regulators and sucrose in the medium increased osmotic stress thereby inducing H2O2 accumulation in the explant cells. We found that H2O2 concentration was significantly decreased in explant cells when the induction medium was modified, i.e., when reducing the concentration of sucrose, which reduces the osmotic pressure of the medium, or by withdrawing plant growth regulators at mid-culture. These treatments also decreased the proportion of explant cells undergoing programmed cell death. Accordingly, a decreased rate of somatic embryo induction was observed. These results show that PCD occurred during tissue browning and death of some explant cells during somatic embryogenesis in F. mandshurica. The ROS contributed to PCD in abiotic stress stimulated F. mandshurica cells.

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 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 the thin cell layer technique in the induction of somatic embryos in Pinus patula Schl. et Cham

Pinus patula is a species commonly used for reforestation in Mexico.However,efficient methods for the mass production seedlings are required.Micropropagation particularly by somatic embryogenesis provides an option for the rapid multiplication of high-quality,genetically improved material.This study induces somatic embryogenesis in this species using the thin cell layer(TCL)technique.Two sources of explants(complete immature embryos;lTCL segments from immature embryos)were evaluated.The efficiency of TCL from longitudinal sections[lTCL]and transverse[tTCL]was evaluated.The results show using thin cell layers from immature embryos cultivated in 16 light/8 dark hours achieves induction of somatic embryos.A higher percentage of embryogenic callus was obtained when tTCL segments were used as an explant source.These results produced somatic embryos from tTCL segments of an immature embryo without germinating the seed,making the process more time efficient.In addition,this technique can be used to generate somatic embryogenesis in forest species that have low germination rates.

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.

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

Morphology and ontogeny of directly differentiating shoot buds and somatic embryos in Santalum album L.

Santalum album L.is a commercially important tree that yields essential oil of high medicinal value.Regeneration research through organogenesis and embryogenesis has been documented but no report depicts comparative ontogeny of directly differentiating shoot buds(SB)and somatic embryos(SE).In the present study aseptic seedling derived hypocotyl segments(HC)and hypocotyl+root junction(HC+R)were used to induce SBs and SEs,respectively.Ontogenic differences between the structures were confirmed using scanning electron microscopy and histological analysis.MS medium containing 6-benzyladenine or BA(2.5μM)produced highest number of direct SB,while MS+BA(7.5μM)proved suitable for higher frequency of SE differentiation.The differentiating structures attained growth when transferred to MS medium containing a combination of BA and anaphthalene acetic acid or silver nitrate(AgNO3).A combination of indole-3-butyric acid and silver nitrate(AgNO3)in half-strength woody plant medium and lesser osmotic concentration(2%sucrose),induced rhizogenesis.

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.

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.

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.

Macronutrients Effect on Secondary Somatic Embryogenesis of Moroccan Cork Oak (<i>Quercus suber</i>L.)

To define the preliminary embryogenesis culture conditions of Moroccan Cork Oak (Quercus suber L.) in secondary propagation systems, secondary embryos formation from primary embryos were analyzed using seven macronutrient medias: (Chalupa) (BTM), Murashige and Skoog (MS), Schenk and Hildebrant (SH), Schenk and Hildebrant with half content macronutrients (SH ?), full Gamborg (G), Margara (N30K) and Woody Plant Media(WPM). Mature primary embryos at cotyledonal stage of 8 - 10 mm, were placed in each culture medium, and supplemented with 30 g/l of glucose and 7 g/l of agar without PGR. The experimental design consisted of a Petri dish containing three embryos explants. Each one of the seven treatments was composed of ten Petri dishes. Mean number of secondary somatic embryos, clusters and new embryogenic formation on clusters were recorded after 8 weeks, and evaluated by statistical analysis. There were no significant differences (p ≤ 0.05) in clusters and new embryos on clusters formation among evaluated media;but mean number of secondary embryos was significantly higher in N30K (4.37 ± 0.48) compared with control media (1.37 ± 0.15). The morphology of secondary embryos grown in the N30K medium exclusively showed the presence of three embryogenic stages: early cotyledonal with translucide aspect, white opaque, or green, and mature embryos. These results indicate that the medium do influence the morphogenic characteristics of produced embryos. Our finding revealed that secondary somatic embryos produced in N30K medium presented better morphogenic potential, with different stages of embryogenic formation.

In Vitro Somatic Embryogenesis in Some Oil Yielding Tropical Tree Species

Somatic embryogenesis was achieved in two oil yielding tropical tree species i.e. Simarouba glauca & Azadirachta indica using immature zygotic embryos as explants on Murashige and Skoog (MS) medium supplemented with 0.5 – 1.5 mg/l benzylaminopurine (BA) and 2.0 - 3.0 mg/l NAA (1-napthaleneacetic acid) or 2, 4-D (2,4-dichlorophenoxyacetic acid) and 3% sucrose. MS medium containing 1.0 mg/l BA and 2.0 mg/l NAA was noted to be the most effective in inducing friable embryogenic callus (FEC) in Simarouba glauca;the number of somatic embryos per culture varied in MS medium supplemented with 1.0 – 1.5 mg/l BA and 1.0 mg/l NAA. In Azadirachta indica, somatic embryos developed on MS medium supplemented with 0.5 mg/l BA and 1.5 – 2.0 mg/l 2,4-D which were in various shapes and sizes after the first subculture on MS medium supplemented with 0.25 mg/l abscisic acid. The somatic embryos which developed shoots were isolated and rooted in 1/2 strength MS medium supplemented with 0.25 mg/l abscisic acid and 2% sucrose. About 25% of embryos germinated within 20 days of culture in case of Simarouba glauca and 62% in Azadirachta indica. The somatic embryo-derived plantlets were transferred to the field after being hardened in the climate controlled hardening chamber.

Optimization of Germinated Conditions for Somatic Embryos in Liriodendron Hybrids

The development of somatic embryos is a key technology in plant breeding. To gain the best cultural conditions,the factors influencing on germination and transplanting survival rate of the somatic embryos in Liriodendron hybrids were optimized,including the development period of somatic embryos,macro-element,Vitamin C,genotypes,plant hormones and transplant medium in this study. The results showed that the optimal development phase of germination was torpedo-shaped embryo,and the base cultural medium of 3/4 MS with vitamin C can keep normal form from somatic embryos. The germination rate of somatic embryo had obvious genotype difference,among which the hybrid 1 × 5002 was the most sensitive( 82. 46%),and the hybrid 1 × 4088 was the most insensitive( 13. 24%). The medium with 0. 1 mg/L of KT + 0. 1 mg/L of IBA can help promote the germination of somatic embryogenesis. In addition,the yellow soil was more suitable than mixed peat soil for transplanting Liriodendron hybrids seedlings. Therefore,the obtainment of the optimal conditions has important guidance for production practices of Liriodendron hybrids.

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.

Cocoa (<i>Theobroma cacao</i>L.) Somatic Embryos Tolerate Some Ice Crystallization during Cryopreservation

Secondary somatic embryos (SSEs) of cocoa, a recalcitrant tropical, seed-producing species, were cryopreserved using a vitrification approach and Differential Scanning Calorimetry (DSC) was employed to optimise sucrose preculture and Plant Vitrification Solution 2 (PVS2) incubation. The objective of the study was to evaluate the influence of sucrose preculture and PVS2 dehydration on water content of SSE that will enable it to survive cryostorage. SSEs were precultured for 3 or 5 days on media containing 0.5 M or 0.75 M sucrose and cryoprotected in loading solution (2 M glycerol and 0.4 M sucrose in medium) for 20 min before they were dehydrated with cold PVS2 for 0 - 90 min. Thermal analysis revealed the occurrence of ice crystallization in the SSEs with the extent declining with increasing PVS2 exposure. Maximal survival of SSEs was promoted by preculture on 0.5 M sucrose medium and dehydration with PVS2 for 45 - 60 min, which was characterised by small ice crystallization. Exposure of SSEs beyond 60 min leads to excessive dehydration as characterized by no change in the thermograms. Based on these findings, preculture of SSEs on 0.5 M sucrose medium and dehydration with cold PVS2 for 60 min has been adopted for the successful cryopreservation of cocoa germplasm.

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.

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).

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.