Somatic Embryogenesis and Plant Regeneration from Two Recalcitrant Genotypes of Gossypium hirsutum L.

An improved protocol has been developed for somatic embryogenesis and plant regeneration of recalcitrant cotton cultivars. High callus frequencies and embryogenic tissue were developed in MSB medium supplemented with gradient concentrations of KT and 2,4-D, their concentration decreasing from 0.1 to 0.01 mg·L^-1. Somatic embryos were successfully incubated in 1/2 macronutrient MSB suspension supplemented with 0.5 g· L^-1 glutamine and 0.5 g·L^-1 asparagine. Decrease in macronutrient concentration of MSB significantly alleviated browning and was beneficial to suspension cells. Transformation of somatic embryos into plants was induced in MSB medium supplemented with 3% sucrose, 0.5 g·L^-1 glutamine, 0.5 g·L^-1 asparagine, and 6.0 g·L^-1 agar. The effect of sucrose as carbohydrate was better than that of glucose for plant germination. Using this protocol, regenerated plantlets from the CCRI521 and Zhongzhi86-6 reached to as much as 19.6 and 18.5% somatic embryos, respectively.

Enhanced Somatic Embryogenesis and Plant Regeneration from Embryogenic Cultures Derived from Mature Loblolly Pine Zygotic Embryos by Suspension Culture and Medium Selection

Mature zygotic embryos of three families of loblolly pine ( Pinus taeda L.) were cultured on callus induction medium containing 8?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 4?mg·L -1 6 benzyladenine (BA), 4?mg·L -1 kinetin (KT), 500?mg·L -1 casein hydrolysate, and 500?mg·L -1 glutamine for 9 weeks, callus was formed on cotyledons, hypocotyls, and radicles of mature zygotic embryos. Callus was sub cultured on the callus proliferation medium with 1 6?mg·L -1 2,4 dichlorophenoxyacetic acid (2,4 D), 0 8?mg·L -1 6 benzyladenine (BA), 0 8?mg·L -1 kinetin (KT) for 9 weeks. White translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESM) and immature somatic embryos was obtained, and the highest frequency of explants forming embryogenic callus was 16 9%. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing embryogenic suspension masses and immature somatic embryos were transferred to medium containing abscisic acid (ABA), polyethylene glycols (PEG), or activated charcoal for enhancing the production of cotyledonary somatic embryos. After mature somatic embryos were cultured on medium containing indole butyric acid (IBA), gibberellic acid (GA 3), BA, and activated charcoal and being lowered sucrose concentration for 4～12 weeks, somatic embryos germinated to form regenerated plantlets. Seventy one regenerated plantlets were transferred to a perlits∶peatmoss∶vermiculate (1∶1∶1) soil mixture, and 23 plantlets survived in the field.

Conservation of Iris bismarckiana Regel （Iridaceae） Using Plant Regeneration via Somatic Embryogenesis

In an attempt to propagate and conserve the rare, showy bulbous plants of Iris bismarckiana, newly recorded to the flora of Jordan and to contribute to the conservation the wild lris species in Jordan, a simple rapid, time consuming protocol has been developed using plant regeneration via somatic embryogenesis. Somatic embryogenesis was induced in zygotic embryo culture on Murashige and Skoog （MS） medium supplemented with 2,4-dichlorophenoxy acetic acid （8 mg/L） as the sole plant growth regulator, where both embryogenesis calli and somatic embryos were induced. Separation of embryos from embryo clusters was necessary to enhance the frequency of germination. Germination was stimulated by separation of embryos successfully from embryo clusters and transferred onto fresh MS medium. Data obtained were analyzed as a complete random design with three replications. Calli fragments that were transferred to embryogenesis induction medium （EIM） produced white embryo-like globular structures within two weeks. Within three more weeks, clusters of structures at various stages of development could be found on the same callus. The applied technique is rewarding and encouraging for further research on the endangered wild species of Iris in Jordan.

Indirect somatic embryogenesis and regeneration of Fraxinus mandshurica plants via callus tissue

Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo(SE) yield,unsynchronized SE development,and a high percentage of deformed SEs.We aimed to improve F.mandshurica SE production by synchronizing SE development,improving SE quality,and inducing root formation to obtain complete regenerated plants.Cotyledons of immature zygotic embryos of F.mandshurica were induced to form callus and then SEs.The SE induction percentage from explants differed among 32 mother trees,and the one with the highest SE induction percentage(29.8%) was used for further experiments.The highest callus induction percentage was94.2% on 1/2-strength Murashige and Skoog medium(MS1/2)supplemented with 0.15 mg·L^(-1) naphthalene acetic acid.The highest callus proliferation coefficient(240.5) was obtained on McCown's Woody Plant Medium containing 0.1 mg·L^(-1)6-benzyl adenine and 0.15 mg·L^(-1) 2,4-dichlorophenoxyacetic acid.The highest number of SEs(1020.5 g~(-1) fresh weight) was obtained on MS1/2 medium supplemented with1 mg·L^(-1) 6-benzyladenine.The highest number of cotyledon embryos(397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L^(-1) abscisic acid and then applying a drying treatment.The cotyledon embryos were milky white,uniformly sized(average length 4.7 mm),and 80% of them were normal.The SE rooting percentage on 1/2 MS medium containing 0.01 mg·L^(-1) NAA was 37.5%.Overall,the germination percentage of SEs was 26.4%,and complete regenerated plants were obtained after transplanting and acclimation.These results provide more possibilities for the preservation and breeding of F.mandshurica.

Plant Regeneration of Sweet Potato via Somatic Embryogenesis from Different Explants

[Objective] This study aimed to regenerate plants of sweet potato (Ipomoea batatas) cultivar Xushu22 via somatic embryogenesis, using leaf and shoot apex as explants. [Method] The leaf and shoot apex of Xushu 22 were separately cultured on MSB medium and MSD medium. The induced embryogenic calluses were then cultured on MS medium. The regeneration frequency of leaf and shoot apex explants were respectively calculated. [Result] The average frequency of leaf explants developing somatic callus was 95.69% compared to 30.56% in case of shoot apex explants. There were different types of morphogenic structures in the process of somatic embryo development. Leaf explants gave a high regeneration frequency to 60.61%, while the regeneration frequency of shoot apices was 22%. In addition, no morphological variations were observed in the regeneration plants. [Conclusion] Leaf explant was better than shoot apices in plant regeneration of Xushu22 via somatic embryogenesis.

Somatic Embryogenesis and Organogenesis for Regeneration of Endangered Multipurpose Desert Plant <i>Leptadenia pyrotechnica</i>Forsk. Decne in the Kingdom of Bahrain

Leptadenia pyrotechnica is an important multipurpose endangered plant in the Kingdom of Bahrain with restricted distribution. Nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of indole acetic acid (IAA) and 6-benzylaminopurine (BAP). Initially, 80% and 60% explants responded in direct shoot and callus initiation response respectively in presence of 8.88 μM BAP with 5.71 μM IAA in modified MS media after two weeks of culture. The highest frequency of plant regeneration was observed in presence of 8.88 μM BAP with 1.14 μM IAA following organogenic pathway of differentiation. Hundred percent callus proliferation was observed while initial callus developed in presence of 4.44 μM BAP with 2.85 μM IAA and was transferred in media containing 4.44 μM, 6.66 μM BAP with 2.85 μM IAA and 13.32 μM BAP with 5.71 μM IAA. The callus derived plants were regenerated following the pathway of indirect somatic embryogenesis. The induction of somatic embryogenesis and plant regeneration from callus was also observed in modified MS media supplemented with 4.44 μM BAP and 2.85 μM IAA. The plant regeneration protocol we developed for Leptadenia pyrotechnica will be very beneficial for biodiversity conservation and environment protection of Bahrain. Moreover, the present paper reports for the first time specifically the somatic embryogenesis in this multipurpose desert plant Leptadenia pyrotechnica.

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

Establishment of Plant Regeneration System from Immature Embryos of Maize(Zea mays L.) Inbred Lines

[Objective] The aim was to explore the conditions of high frequency induction of embryonic callus and plant regeneration of maize. [Method] Immature embryos of maize inbred lines were used as explants to study the effect of different 2,4-D concentrations on the induction of callus,the effect of different 6-BA concentrations on the differentiation of test-tube plantlet,as well as the effect of different IBA concentrations on the rooting of test-tube plantlet. [Result] 2,4-D showed obvious effect on the induction of inducement rate of maize,and the optimum induction medium was：N6 ＋ 2 mg/L of 2,4-D ＋ 500 mg/L of CH ＋ 500 mg/L of Pro ＋10 mg/L of AgNO3; the optimum differentiation medium was：N6 ＋ 0.5 mg/L of BA ＋ 500 mg/L of Pro; the optimum for the rooting of test-tube plantlet was 1/2 MS ＋ 0.5 mg/L of IBA. [Conclusion] The study had provided basis for the genetic transformation of maize.

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.

Study on Plant Regeneration of Wheat Mature Embryos Under Endosperm-Supported Culture

To reveal the suitability of using mature embryos as an explant source in wheat tissue culture, mature embryos from eight common wheat cultivars （Triticum aestivum L. cv.） were cultured with or without endosperm to test their efficiency of callus induction and plant regeneration. When embryos were cultured together with endosperm （endosperm-supported culture, ES）, the percentage of callus induction was significantly lower than that when embryos were cultured in the absence of endosperm （non-endosperm-supported culture, NES）. This pattern was evident in most genotypes, regardless of whether 2 or 8 mg L^-1 2,4-D was added in the NES culture. However, in ES culture, more induced calli were differentiated into distinct green spots and they further developed into plantlets. Thus, more plants were regenerated in ES culture than in the NES treatment. Most of the eight tested genotypes showed a significant difference in callus induction rate and plantlet regeneration in both ES and NES cultures. In addition, the enzymatic activity of oxalate oxidase in the callus of ES culture condition was obviously higher than that in the callus of NES culture condition, suggesting that the activity of oxalate oxidase may be a parameter for selection of calli with potential for plantlet regeneration. These results indicate that wheat mature embryos are valuable explants for highly efficient callus induction and plant regeneration, if proper treatment and medium are used.

Dicamba and Sugar Effects on Callus Induction and Plant Regeneration from Mature Embryo Culture of Wheat

To establish a highly efficient plant regeneration system for wheat genetic transformation, the effects of three different concentrations of dicamba and two different sugar types on callus induction and plant regeneration from mature embryo cultures were evaluated. Callus induction and plant regeneration were obtained from mature embryos of two commercial cultivars Zhoumai 18 and Yumai 34 （Triticum aestivum L.） cultured on L3 basal medium. The results showed that the efficiency of mature embryo culture was significantly influenced by the genotypes, sugar types and dicamba concentrations. 4 mg L^-1 dicamba proved the best effective for inducing embryogenic callus and also gave the highest proportion of plants regenerated across the two cultivars. Substitution of maltose by sucrose significantly improved the plant regeneration efficiency in both cultivars. There was a significant interaction between genotype-by-sugar types, and sugar types-bydicamba concentrations. Overall, Zhoumai 18 gave the highest frequency of plant regeneration （82.65%） when dicamba concentration was 4.0 mg L^-1 and with sucrose in initial callus induction. These results will facilitate genetic transformation work with elite wheat.

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.

Advancements in plant regeneration and genetic transformation of grapevine(Vitis spp.)

Grapevine(Vitis spp.)is one of the most economically important fruit crops worldwide,and there is considerable interest in improving its major agronomic and enological traits in response to ever-changing agricultural environments and consumer demands.Molecular genetic techniques in particular,associated with rapid technological advancements,provide an attractive alternative to conventional breeding approaches for developing new grapevine varieties with enhanced yield performance,quality,stress tolerance and disease resistance.To date,several grapevine varieties have been transformed with genes associated with diverse functions through biolistic bombardment and/or Agrobacterium-mediated transformation,and transgenic grape lines have been obtained using established regeneration systems.Nevertheless,a wide range of factors,including genotype,explant source and culture medium,have been shown to affect the efficiency of plant regeneration.Moreover,the selection and use of acceptor materials,bacterial strain and cell density,selectable markers and selection methods also influence transformation efficiency.This paper provides an overview of recent advances in grapevine regeneration and genetic transformation and in-depth discussion of the major limiting factors,and discusses promising future strategies to develop robust plant regeneration and genetic transformation in grapevine.

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.

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.

Establishment of highly efficient plant regeneration of Paeonia ostii‘Fengdan’through optimization of callus,adventitious shoot,and rooting induction

Tree peony is a famous ornamental plant,while the low propagation rate is the main hurdles hindering the industry development.Till now,the highly efficient regeneration system for tree peony is not established.In this study,using Paeonia ostii’Fengdan’mature embryos,the effects of variations in inoculation method,initiating culture,adventitious shoot induction,rooting media,plant growth regulators(PGRs),and a nonconventional PGR(plant extracts)on regeneration from explants were evaluated.In embryo cultures,embryonic callus induction rate of 1/4 embryos was the highest among those of embryos with other three technical treatments(whole embryos,1/2 embryos,and pieces of embryos).The woody plant medium(WPM)containing 1.0 mg·L^(-1)6-BA,0.5 mg·L^(-1)GA3,30.0 g·L^(-1)sucrose,and 3.0 g·L^(-1)phytagel significantly improved shoot induction and multiplication.3.0 mg·L^(-1)plant extracts promoted hypocotyl germination,rooting,and root growth,in direct embryo culture,and a combination of 3.0 mg·L^(-1)plant extracts+2.0 mg·L^(-1)IBA+1.5 mg·L^(-1)IAA produced optimal rooting induction rate for multiple shoots in direct embryo culture and indirect somatic embryogenesis.For the three in vitro micropropagation methods,the highest shoot proliferation coefficient(5.4±0.2)was obtained with indirect somatic embryogenesis.Fortunately,the propagation ability of shoots remains high,even when culture propagation was continued for more than two years.Thus,a reliable system for plant regeneration from mature embryos derived from P.ostii’Fengdan’callus and two direct embryo culture systems have been established.The novel regeneration system could facilitate uniform seedling production.

Direct somatic embryogenesis from leaves,cotyledons and hypocotyls of Hippophae rhamnoides

Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in Hippophae rhamnoides L. was achieved. The influence of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants was studied. The highest frequency of somatic embryo production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg/L kinetin (KIN) and 0.2, 0.5 mg/L indole-3-butyric acid (IAA). Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55%. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.

Efficient Plant Regeneration with Arabinogalactan-Proteins on Various Ploidy Levels of Cereals

To determine the most effective dose of arabinogalactan-protein （AGP） in regeneration medium, mature embryos of genotypes in three different ploidy levels （Triticum aestivum L. cv. Ikizce-96, Triticum durum Desf. cv. Mirzabey and Hordeum vulgare L. cv. Tokak） were used to establish an efficient plant regeneration system for cereals. Percentage of callus production, capacity of regeneration were calculated, and also culture effect, root, stem, and total plant length of regenerant plants were observed in six different regeneration media （MS control, MS＋2, 5, 7, 10, 12 mg L-1 AGP） in these three different genotypes. According to the results, the highest amount of callus production was found in Ikizce-96 as 93.75% using 5 mg L-1 dicamba and 1 mg L-1 kinetin in induction medium. However, the most improved callus was observed in diploid barley Tokak as 179.95 mg in weight and 6.18 mm in diameter, respectively. The highest regeneration capacity was observed in the dose of 5 mg L-1 AGP in MS of all the genotypes and hexaploid wheat Ikizce-96 gave the best results with the highest regeneration capacity and culture effects （94.86 and 92.5%） in the same dose of AGE These results indicated that effective dose of AGP in regeneration medium increase plant regeneration in calli derived from cereal mature embryos.

Plant Regeneration through Somatic Embryo in Herpetospermum pedunculosum, an Endangered Tibetan Medicinal Herb

Objective An effective reproducible protocol for complete plant regeneration via somatic embryogenesis has been developed for Herpetospermum pedunculosum, an endangered Tibetan medicinal herb. Methods The cotyledon explants used in this study were excised from seedlings germinated in vitro. Callus was induced from cotyledon explants on Murashige and Skoog’s medium, supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.1–1.0 mg/L) alone or in combination with 6-benzylaminopurine (BA, 0.5, 1.0, and 2.0 mg/L). Results The calli showed differentiation of globular embryos after three weeks of incubation on MS medium supplemented with various combinations of BA and NAA. Sixty-two percent of the embryogenic calli produced somatic embryos in MS basal medium supplemented with BA (1.0 mg/L) + NAA (2.0 mg/L). The addition of KN (0.5 mg/L) to MS medium containing both BA and NAA (2.0 mg/L each) significantly increased the frequency of somatic embryogenesis. The maximum percentage of embryogenic calli formation was 83%, and globular embryos formed and germinated successfully in this medium. Then, transferring the regenerated plants from this medium to hormone-free MS medium will further enhanced the development of the plants, and the healthy plantlets are formed successfully within four weeks. The plantlets were transferred to soil to acclimatize under greenhouse conditions and 75% survived. Conclusion Somatic embryogenesis protocol as reported here can play a key role in the propagation and conservation of this endangered species.