Phenolic compound profiling and antioxidant potential of different types of Schisandra henryi in vitro cultures.

Schisandra henryi is an endemic species of medicinal potential known from traditional Chinese medicine. As part of this study, a complex biotechnological and phytochemical assessment was conducted on S. henryi with a focus on phenolic compounds and antioxidant profiling. The following in vitro cultures were tested: microshoot agar and callus, microshoot agitated, and suspension, along with the microshoot culture in PlantForm bioreactors. Qualitative profiling was performed by ultra-high-performance liquid chromatography with a photodiode array detector coupled with ion-trap mass spectrophotometry with electrospray ionization and then quantitative analysis by high-performance liquid chromatography with a diode array detector using standards. In the extracts, mainly the compounds from procyanidins were identified as well as phenolic acids (neochlorogenic acid, caffeic acid, protocatechuic acid) and catechin. The highest content of phenolic compounds was found for in vitro agar microshoot culture (max. total content 229.87 mg/100 g DW) and agitated culture (max. total content 22.82 mg/100 g DW). The max. TPC measured using the Folin-Ciocalteu assay was equal to 1240.51 mg GAE/100 g DW (agar microshoot culture). The extracts were evaluated for their antioxidant potential by the DPPH, FRAP, and chelate iron ion assays. The highest potential was indicated for agar microshoot culture (90% of inhibition and 59.31 nM/L TEAC, respectively). The research conducted on the polyphenol profiling and antioxidant potential of S. henryi in vitro culture extracts indicates the high therapeutic potential of this species. KEY POINTS: • Different types of S. henryi in vitro cultures were compared for the first time. • The S. henryi in vitro culture strong antioxidant potential was determined for the first time. • The polyphenol profiling of different types of S. henryi in vitro cultures was shown.

Synthetic consortia of four strains promote Schisandra chinensis growth by regulating soil microbial community and improving soil fertility.

MAIN CONCLUSION: Synthetic consortia performed better in promoting Schisandra chinensis growth than individual strains, and this result provides valuable information for the development of synthetic microbial fertilizers. Schisandra chinensis is an herbal medicine that can treat numerous diseases. However, the excessive reliance on chemical fertilizers during the plantation of S. chinensis has severely restricted the development of the S. chinensis planting industry. Plant growth-promoting rhizobacteria (PGPR) can promote the growth of a wide range of crops, and synthetic consortia of them are frequently superior to those of a single strain. In this study, we compared the effects of four PGPR and their synthetic consortia on S. chinensis growth. The pot experiment showed that compared with the control, synthetic consortia significantly increased the plant height, biomass, and total chlorophyll contents of S. chinensis, and their combined effects were better than those of individual strains. In addition, they improved the rhizosphere soil fertility (e.g., TC and TN contents) and enzyme activities (e.g., soil urease activity) and affected the composition and structure of soil microbial community significantly, including promoting the enrichment of beneficial microorganisms (e.g., Actinobacteria and Verrucomicrobiota) and increasing the relative abundance of Proteobacteria, a dominant bacterial phylum. They also enhanced the synergistic effect between the soil microorganisms. The correlation analysis between soil physicochemical properties and microbiome revealed that soil microorganisms participated in regulating soil fertility and promoting S. chinensis growth. This study may provide a theoretical basis for the development of synthetic microbial fertilizers for S. chinensis.

Phenolic acid and flavonoid production in agar, agitated and bioreactor-grown microshoot cultures of Schisandra chinensis cv. Sadova No. 1 - a valuable medicinal plant.

In vitro cultures and raw materials (fruits and leaves) of the valuable medicinal plant species - Schisandra chinensis cultivar Sadova No. 1 (SchS) - were evaluated for the production of two groups of phenolic compounds, phenolic acids and flavonoids, and their antioxidant potential. A series of experiments was conducted, aimed at optimizing culture conditions for maximum growth and phenolic production in SchS microshoots. Different concentrations of plant growth regulators (6-benzyladenine - BA and 1-naphthaleneacetic acid - NAA, from 0 to 3 mg/l) in Murashige-Skoog (MS) medium were tested in several cultivation systems (agar, agitated, bioreactor) over various growth periods (10, 20, 30, 40, 50 and 60 days). Furthermore, an elicitation experiment was conducted in which the bioreactor-grown microshoots were exposed to yeast extract. HPLC-DAD analyses confirmed the presence of eight phenolic acids - chlorogenic, cryptochlorogenic, gallic, neochlorogenic, protocatechuic, salicylic, syringic and vanillic, and two flavonoids: kaempferol and quercitrin, in the in vitro biomasses. The highest total phenolic acid (357.93 mg/100 g DW) and flavonoid (105.07 mg/100 g DW) contents were obtained in agar culture extracts cultivated for 30 days on MS medium containing 2 mg/l BA and 0.5 mg/l NAA and for 50 days on MS medium containing 0.1 mg/l BA and 2 mg/l NAA, respectively. These amounts were 1.59- and 5.95-fold lower than in parent plant leaf extracts (569.66 mg/100 g DW), and 4.30- and 1.25-fold higher than in fruit extracts (83.17 mg/100 g DW), respectively. Microshoots grown in a Plantform bioreactor also proved to be a good source of phenolic compounds, however, the elicitor treatment had no noticeable effect on their accumulation. Antioxidant capacity assessed by the Folin-Ciocalteu, FRAP, DPPH and CUPRAC assays revealed significantly higher potential in extracts from in vitro biomass and leaves of the parent plant, as compared to the parent plant fruit extracts.

Schisandra lignans production regulated by different bioreactor type.

Schisandra chinensis (Chinese magnolia vine) is a rich source of therapeutically relevant dibenzocyclooctadiene lignans with anticancer, immunostimulant and hepatoprotective activities. In this work, shoot cultures of S. chinensis were grown in different types of bioreactors with the aim to select a system suitable for the large scale in vitro production of schisandra lignans. The cultures were maintained in Murashige-Skoog (MS) medium supplemented with 3mg/l 6-benzylaminopurine (BA) and 1mg/l 1-naphthaleneacetic acid (NAA). Five bioreactors differing with respect to cultivation mode were tested: two liquid-phase systems (baloon-type bioreactor and bubble-column bioreactor with biomass immobilization), the gas-phase spray bioreactor and two commercially available temporary immersion systems: RITA® and Plantform. The experiments were run for 30 and 60 days in batch mode. The harvested shoots were evaluated for growth and lignan content determined by LC-DAD and LC-DAD-ESI-MS. Of the tested bioreactors, temporary immersion systems provided the best results with respect to biomass production and lignan accumulation: RITA® bioreactor yielded 17.86g/l (dry weight) during 60 day growth period whereas shoots grown for 30 days in Plantform bioreactor contained the highest amount of lignans (546.98mg/100g dry weight), with schisandrin, deoxyschisandrin and gomisin A as the major constituents (118.59, 77.66 and 67.86mg/100g dry weight, respectively).

Extraction and Separation of Active Ingredients in Schisandra chinensis (Turcz.) Baill and the Study of their Antifungal Effects.

Schisandra chinensis extracts (SEs) have traditionally been used as an oriental medicine for the treatment of various human diseases, however, their further application in the biocontrol of plant disease remains poorly understood. This study was conducted to develop eco-friendly botanical pesticides from extracts of S. chinensis and assess whether they could play a key role in plant disease defense. Concentrated active fractions (SE-I, SE-II, and SE-III) were obtained from S. chinensis via specific extraction and separation. Then, lignan-like substances, such as Schisanhenol B, were detected via High-Performance Liquid Chromatography-ElectroSpray Ionization-Mass Spectrometry (HPLC-ESI-MS) analyses of the active fractions. Moreover, the results from biological tests on colony growth inhibition and spore germination indicated that SE-I, SE-II, and SE-III could inhibit hyphal growth and spore generation of three important plant pathogenic fungi (Monilinia fructicola, Fusarium oxysporum, and Botryosphaeria dothidea). The study of the mechanisms of resistant fungi revealed that the oxidation resistance system, including reactive oxygen species (ROS), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD), was activated. The expression of genes related to defense, such as pathogenesis-related protein (PR4), α-farnesene synthase (AFS), polyphenol oxidase (PPO), and phenylalanine ammonia lyase (PAL) were shown to be up-regulated after treatment with SEs, which suggested an increase in apple immunity and that fruits were induced to effectively defend against the infection of pathogenic fungi (B. dothidea). This study revealed that SEs and their lignans represent promising resources for the development of safe, effective, and multi-targeted agents against pathogenic fungi.

Accumulation of dibenzocyclooctadiene lignans in agar cultures and in stationary and agitated liquid cultures of Schisandra chinensis (Turcz.) Baill.

Schisandra chinensis plant in vitro cultures were maintained on Murashige and Skoog (MS) medium supplemented with 3 mg/l 6-benzyladenine (BA) and 1 mg/l 1-naphthaleneacetic acid (NAA) in an agar system and also in two different liquid systems: stationary and agitated. Liquid cultures were grown in batch (30 and 60 days) and fed-batch modes. In the methanolic extracts from lyophilized biomasses and in the media, quantification of fourteen dibenzocyclooctadiene lignans identified based on co-chromatography with authentic standards using high-performance liquid chromatography with diode array detection (HPLC-DAD) and/or liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI-MS) methods. For comparison purposes, phytochemical analyses were performed of lignans in the leaves and fruits of the parent plant. The main lignans detected in the biomass extracts from all the tested systems were schisandrin (max. 65.62 mg/100 g dry weight (DW)), angeloyl-/tigloylgomisin Q (max. 49.73 mg/100 g DW), deoxyschisandrin (max. 43.65 mg/100 g DW), and gomisin A (max. 34.36 mg/100 g DW). The highest total amounts of lignans in the two tested stationary systems were found in extracts from the biomass harvested after 30 days of batch cultivation: 237.86 mg/100 g DW and 274.65 mg/100 g DW, respectively. In the agitated culture, the total content reached a maximum value of 244.80 mg/100 g DW after 60 days of the fed-batch mode of cultivation. The lignans were not detected in the media. This is the first report which documents the potential usefulness of S. chinensis shoot cultures cultivated in liquid systems for practical purposes.

[Quality assessment on schisandrae fructus from different habitats].

OBJECTIVE: To assay five lignans in Schisandrae Fructus collected from different habitats. METHODS: HPLC method was developed to assay Schizandrol A, Schizandrol B, Schisantherain A, deoxyschizandrin and gamma-schizandrin in Schisandrae Fructus. Hierarchical clustering analysis (HCA) and principal components analysis (PCA) were performed to evaluate and classify 30 batches of samples based on the contents of the five lignans using SOLO and SPSS 19.0 software. RESULTS: 30 batches of samples were divided into three groups, which reflecting their quality characteristics. CONCLUSION: PCA and HCA provide the basis for the classification and quality evaluation of Schisandrae Fructus. The content analysis provides reference for resources saving and rational using of Schisandrae Fructus.

An improve method for somatic embryogenesis of Schisandra chinensis (Turcz.) Baillon.

A somatic embryogenesis protocol for plant regeneration of Schisandra chinensis (Turcz.) Baillon was established. Increased callus induction was obtained from mature zygotic embryos on Murashige and Skoog (MS) medium supplemented with 1.0 mg L(-1) N-phenyl-1,2,3-thidiazol-5-yl-urea (TDZ) or 2.0 mg L(-1) 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of Zeatin (Zt) promoted the formation of embryogenic calli. To induce somatic embryogenesis, 2,4-D, TDZ and Zt were incorporated in the medium alone or in combination. Development of the maximum number of somatic embryos (81 globular, 37 heart, 52 torpedo and 37 cotyledon-stage) and germination of the highest number of embryos (50%) was observed on 1/2 MS medium supplemented with 1.0 mg L(-1) TDZ and 0.2 mg L(-1) Zt. Further development of somatic embryos into plantlets was completed in 1/2 MS medium free of plant growth regulators.

Intercarpellary growth of pollen tubes in the extragynoecial compitum and its contribution to fruit set in an apocarpous species, Schisandra sphenanthera (Schisandraceae).

PREMISE OF THE STUDY: Apocarpous plants possess carpels that are separated in the gynoecium. Extragynoecial compita, commonly occurring in basal angiosperms, have been proposed to have the potential to increase offspring quantity in apocarpous species through the intercarpellary growth of pollen tubes. To date, the impact of an extragynoecial compitum on fruit or seed set has not been studied in any species. This study investigated the pollen tube pathway between adjacent carpels and its contribution to fruit set in Schisandra sphenanthera. METHODS: We investigated the fruit set ratio in the field and collected hundreds of gynoecia at their full flowering stage. Pollinated carpel ratio and pollen tube pathway observations were performed using fluorescence optics. KEY RESULTS: Pollen grains germinated and tubes extended along the pseudostyle surface. Some of them turned and entered the ovules at the end of the stigmatic crest, whereas others subsequently grew into neighboring carpels through promontory connections located at the base of the unfused carpels. No tubes were found growing on the surface of the receptacle. More than 24 carpels could be fertilized by pollen tubes from one carpel through hand pollination. The pollinated carpel ratio was significantly lower than the fruit set ratio under natural conditions. CONCLUSIONS: Pollen tubes from one carpel can easily cross in the extragynoecial compitum between the adjacent carpels of S. sphenanthera, and this intercarpellary growth of pollen tubes can significantly increase the fruit set of apocarpous species, at least in S. sphenanthera.

In vitro cultures of Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine)--a potential biotechnological rich source of therapeutically important phenolic acids.

The contents of free phenolic acids and cinnamic acid were determined using an HPLC method in methanolic extracts from biomass of Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine) at different stages of organogenesis, cultured in vitro on a few variants of Murashige and Skoog (MS) medium, containing different concentrations of plant growth regulators 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) (from 0.1 to 3.0 mg/l) and in extracts from overground parts of plants growing in vivo. Six of 12 analysed compounds were detected in all extracts: chlorogenic, p-coumaric, p-hydroxybenzoic, protocatechuic, salicylic and syringic acids. Total contents of the examined metabolites in biomass of shoot-differentiating callus culture cultivated on six MS medium variants were dependent on concentrations of growth regulators in the media and ranged from 14.90 to 60.05 mg/100 g d.w. Total contents of the compounds in biomass extracts from undifferentiating callus culture maintained only on two of six MS medium variants were higher and amounted to 74.54 and 78.24 mg/100 g d.w. Maximum total contents of phenolic acids in both types of in vitro cultures were greater than in fruits (55.73 mg/100 g d.w.) and leaves (4.55 mg/100 g d.w.) of plants gowning in vivo. Chlorogenic acid and salicylic acid were the main compounds identified in biomass extracts of shoot-differentiating callus cultures (max 22.60 and 21.17 mg/100 g d.w., respectively), while chlorogenic acid (max 38.43 mg/100 g d.w.) and protocatechuic acid (max 20.95 mg/100 g d.w.) prevailed in the extracts from undifferentiating callus cultures. Other compounds dominated in fruits, namely p-coumaric acid (23.36 mg/100 g d.w.) and syringic acid (14.96 mg/100 g d.w.). This is the first report on biochemical potential of cells from S. chinensis in vitro cultures to produce the biologically active phenolic acids. These are the first results on the analysis of this group of metabolites in overground parts of plants growing in vivo, too.

[Current distribution of Schisandra chinensis in China and its predicted responses to climate change].

With integration of literature data, specimens records, and field surveys, the current distribution map of Schisandra chinensis in China was drawn, and, based on this map and considering 21 environmental factors, the future distribution of S. chinensis in China in the 2050s and 2080s under the IPCC A2 and A1B climate change scenarios was predicted by using Maxent software. Currently, the S. chinensis in China occurred in 15 provinces, involving 151 counties, and its distribution area decreased with decreasing latitude and longitude. The main distribution area included Heilongjiang, Liaoning, Inner Mongolia, and Jilin. The potential distribution area of S. chinensis in China was 145.12 x 10(4) km2, 48.6% of which were the favorable habitat area, mainly distributed in Changbai Mountains, Xiaoxing'anling Mountains, Daxing'anling Mountains, and the regions between Hebei and Liaoning provinces. The most favorable habitat area only accounted for 0.3%, and was mainly in the Kuandian Manchu Autonomous County, Benxi Manchu Autonomous County, and Huanren Manchu Autonomous County of Liaoning Province, the Antu County and Helong County of Jilin Province, and the Yakeshi City of Inner Mongolia. Under the two climate change scenarios, the potential future distribution area of S. chinensis in China would have a gradual decrease, and the decrement would be larger under A2 than under A1B scenario. By 2050, the distribution area of the S. chinensis under A1B and A2 scenarios would be moderately decreased to 84.0% and 81.5% of the current distribution area, respectively; by 2080, the distribution of S. chinensis under A2 scenario would be dramatically decreased to only 0.5% of the current range, and that under A1B scenario would be decreased to 1/2 of the current range.

[Quality evaluation and antioxidant activity research of Schisandra chinensis from various habitats].

OBJECTIVE: To determine the contents of lignans, crude polysaccharides (CP) and total phenolic compounds (TPC) of Schisandra chinensis from various habitats in Liaoning province and evaluate their quality and free radical scavenging (FRS) activity. METHODS: Contents of schisandrol, deoxyschizandrin and schisandrin B were determined by RP-HPLC. Contents of TPC, CP and FRS activity were determined by Folin-Cicalteu's, phenol-sulfuric acid and DPPH x method, respectively. RESULTS: Sample from Liaoyang city had the highest contents of lignans (21.75 mg/g); Sample from Shenbei New district of Shenyang city had the highest contents of CP (88.72 mg/g); Sample from Guanmenshan district of Benxi city had the highest contents of TPC and FRS activity (26.06 mg/g and 86.3%, respectively). Linear regression analysis results showed that contents of TPC had higher correlation coefficient with FRS activity than that of lignans. Their linear regression equations were Y = 1.3677X + 46.97, R2 = 0.6869 and Y = 2.5916X + 57.927, R2 = 0.1747 for TPC and lignans with FRS activity, respectively. CONCLUSION: The contents of lignans, CP and TPC are significantly different from samples collected from various habitats in Liaoning province. The main antioxidative substances are TPC, and lignans have no significant correlation with FRS activity.

[Influence of different processing method and mature stage on main chemical constituents of Schisandra chinensis].

OBJECTIVE: To study different processing method and mature stage on the quantity of the Schisandra chinensis in the Xinbin region of Liaoning. METHOD: The schisantherin A and 5-hydroxymethylufrurfal (5-HMF) content in S. chinensis was determined by using the HPLC method. The organic acids content was determined by potentiometric titration. The polysaccharide content was determined by oxidation reduction titration. The volatile oil content was determined by steam distillation. RESULT: The quantity of S. chinensis was different due to different processing method and mature stage. CONCLUSION: The different processing method and mature stage have significant influence on the quantity of S. chinensis.

Differentiation of various kinds of Fructus schisandrae by surface desorption atmospheric pressure chemical ionization mass spectrometry combined with principal component analysis.

Various kinds of Fructus schisandrae were studied by surface desorption atmospheric pressure chemical ionization mass spectrometry (DAPCI-MS) without any sample pretreatment. The volatile components in F. schisandrae were detected in the ambient environment and the analytical time for each sample was only 30s. F. schisandrae are produced mainly in 5 different geographical regions (Elunchun, Mudanjiang, Tonghua, Tieling and Shangluo), and they could be successfully differentiated according to their chemical markers by Principal Component Analysis (PCA). A total of 8 components which gave more contribution for PCA analysis were unambiguously identified by comparison of the MS(2) data of chemical markers to the data of reference compounds as reported in the literature. Similarly, wild grown and cultivatable species of F. schisandrae were well separated by the above-mentioned method. In addition, raw and processed cultivatable F. schisandrae (steamed by water, alcohol, vinegar, or honey, and fried by honey) were found to be clustered at different location, respectively. Furthermore, the clustered degree of differently processed products was correlated with their clinical effects. Our results demonstrated that DAPCI-MS in combination with PCA was a feasible technique for high-throughput differentiation of various kinds of F. schisandrae. It is also possible that DAPCI-MS could become a powerful technology in the studies of traditional Chinese medicine studies and in situ analysis of Chinese herbs.

The compositions of volatiles and aroma-active compounds in dried omija fruits (Schisandra chinensis Baillon) according to the cultivation areas.

Differences in the compositions of volatiles from dried omija fruits (Schisandra chinensis Baillon) cultivated in different areas (Mungyeong, Jangsu, Jechon, and Hoengseong) in South Korea were determined by applying principal component analysis to gas chromatography-mass spectrometry data sets. Quantitative assessments revealed that terpene hydrocarbons, such as germacrene D, ß-selinene, α-ylangene, ß-elemene, α-selinene, and (E)-ß-farnesene, were the main volatiles in all omija fruit samples. On the other hand, (E)-ß-ocimene, calarene, (E)-ß-farnesene, ß-selinene, nonanal, 2-methylbutanoic acid, benzoic acid, 2,3-butanediol, and phenethyl alcohol were the major volatile components that contributed to the discrimination between omija fruit samples from the four cultivation areas. In addition, aroma-active compounds in four dried omija fruits were investigated and compared by gas chromatography-olfactometry using aroma extract dilution analysis. (E)-ß-Ocimene (floral and herbaceous), α-pinene (pine-like and woody), hexanal (cut grass-like), 5-methylfurfural (burnt sugar-like and sweet), and α-terpinene (minty, green, and fresh) were important aroma-active compounds in all omija samples. Interestingly, the flavor dilution factors of most aroma-active compounds were lower for omija sample cultivated in Hoengseong than for those cultivated in Mungyeong, Jangsu, and Jechon.

[Quality evaluation of Schisandra chinensis at different producing areas in northeast China].

OBJECTIVE: To investigate the quality of Schisandra chinensis, and thus provide the useful reference data for its quality standard establishment and standardized cultivation. METHOD: Base on the Chinese Pharmacopoeia and literature relating to S. chinensis, the appearance, water-soluble extractive, total ash, acid-insoluble ash and volatile oil of 10 S. chinensis sampes collected from different producing sites in northeast China were studied or determined, and the contents of major active lingans were measured by HPLC. RESULT: The content of total ash and acid-insoluble ash in S. chinensis was less than 7.0%, 1.0%, respectively. The contents of water-soluble extractive were higher than 45.0%. HPLC analyses showed that the content of schisandrol A was higher than 0.40%. All these results in accordance with the requirements of Chinese Pharmacopoeia were published in 2010. CONCLUSION: The differences of the appearance and the internal quality are different for the samples collected from different areas. And the establishment of a scientific and comprehensive quality standard system will be very important role in ensuring the quality of S. chinensis.

[Response of net photosyntheitc rate to light and CO2 in Schisandra chinensis from different region and growing situation].

American CI-340 portable photosynthesis system was applied to compare the response of the net photosyntheitc rate to the light and the CO2 in Schisandra chinensis form different region and growing situation. The result showed the sample plant from Liaoning had higher light compensate point, higher light saturation point, higher maximum Pn value and higher apparent quantum yield than the sample from Jilin, so it can adapt to the changes of the sunlight in a day better. The weak plant from Jilin had lower light compenstate point, higher light saturation point and higher net photosynthetic rate, so it had stronger availability on light. The stronger one was more sensitive to the weak light. The Jilin sample had higher CE and lower CO2 compensate point compared to that from Liaoning, but when the density of CO2 rised to 240 micromol/mol, the Pn of Schisandra chinensis in Liaoning became much higher than that of Jilin. Under the natural CO2 density condition, the plant from Liaoning had higher photosynthesis ability.

[The changes of three endogenous hormones during flower bud differentiation of Schisandga chinensis].

OBJECTIVE: To reveal the relation between endogenous hormones and the flower bud differentiation in Schisandga chinensis. METHOD: Top buds of extremely short branch and axillary buds of long branch in the same plant of S. chinensis were used as material and the contents of endogenous hormones were measured during different periods of the flower bud differentiation with HPLC. RESULT: The result showed that flower bud differentiation and the formation of female flower were inhibited by high concentration of GA3 and were promoted by high concentration of ABA or ZT. Low ratio of GA3/ABA has the same result. CONCLUSION: There was a correlation between endogenous hormones and the flower bud differentiation of S. chinensis.

[Seed germinative characteristics and embryo development of Schisandra chinensis (Turcz.) Bail].

OBJECTIVE: To detect the germinative character of S. chinensis seeds. METHOD: Using low temperature and gibberellin treatment. RESULT: The breach rate of seeds was increased by both low temperature and gibberellin, but the breach on different seeds appeared untidy, which showing a character. The embryo development is normal before breaching and increased in volume rapidly after breaching. CONCLUSION: The bypoplasia of embryos is not the primary reason for long dormancy of the seeds.