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 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.

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.

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.

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.

Somatic Embryogenesis and Plantlet Regeneration in Callus Cultures Derived from Mature Zygotic Embryos of Slash Pine

White, translucent, and mucilaginous embryogenic callus was initiated in cultured mature zygotic embryoexplants of two different seed sources of slash pine(Pinus elliottii) on several culture media containing auxin and cytokinin.Somatic proembryos were induced on media containing 2,4-D and BA. Maturatiol1 was successfully achieved on mediumsupplemented with ABA. Somatic embryos germinated into regeneration plantlets On DCR medium containing activated charcoal. HiStological observatiol1 and scanning electron inicroscopic observatiol1 showed that proembryos derived from em-bryonal suspensor mass(ESM) were formed on the surface or the inside of embryogenic callus, and the prolitbration of pro-embryos was mainly from clcavage polyembryos.

Effect of Gibberellin,Light and Genotype on Somatic Embryogenesis and Plantlet Regeneration of Peanut

[ Objective] Peanut in vitro regeneration system was optimized to provide technical support for its genetic transformation and mutagenesis. [ Method ] The effect of gibberellin, light and genotype on somatic embryogenesis and plantiet regeneration of peanut was studied using Guihua peanut cuhivar as materials, leallets as explants, MS ＋ 10 mg/L 2, 4-D as somatic embryo induction medium, and MS ＋3 mg/L 6-BA ＋0. 8 mg/L NAA ＋ （0 - 15 mg/L） GA3 as plantlet induction me- dium. [Result]The somatic embryo induction rate of five peanut varieties under light condition of light： dark = 14 h： 10 h was significantly higher compared with that under dark condition by 7, 5 - 37.5 percent points. Among different peanut varieties, Guihua 26 represented the highest somatic embryo induction rate of 62. 8%, while Guihua 833 represented the lowest somatic embryo induction rate of 21.7%. Adding 5 - 15 mg/LGA3 in plantlet induction medium was conducive to improving the induction rate of plandets derived from somatic embryos. With addition of 5 mg/L GA3, Guihua 26 and Guihna 771 represented the highest plantlet induction rate of 42.8% and 35.3%, respectively; with addition of 15 mg/L GA3, Guihua 836, Guihua 1026 and Guihna 833 represented the highest plantlet in- duction rate of 38.7%, 33.3% and 26.4L%, respectively. Among different combinations of peanut variety and GA3 concentration, plantlet induction rate reached the highest in the combination Guihua 26 ＋ 5 mg/L CA.3 arid reached the lowest in the combination Guihua 836 ＋ 15 mg/L GA3. [ Conclusion] Appropriate light conditions are conducive to peanut somatic embryogenesis in vitro. Adding GA3 in plantlet induction medium is conducive to promoting plantlet regeneration. Guihua 26 and Guihua 836 are the best peanut varieties among the experimental genotypes for somatic embryogenesis and plant regeneration.

<i>In Vitro</i>Regeneration of <i>Curcuma caesia</i>Plantlets from Basal Part and via Somatic Embryogenesis

Plantlets of Curcuma caesia were produced in vitro from newly sprouting vegetative buds of tubers. Segments of the plantlets from the junction between the root and the basal portion of the stem were subsequently used as explants to investigate factors affecting callus induction and plant regeneration via somatic embryogenesis. The explants were placed on Woody Plant Medium (WPM) together with different concentrations of 2,4-dichlorophenoxyacetic acid(2,4-D) and benzyl aminopurine (BAP) in the presence of light. The growth medium supplemented with 5 mg/L BAP and 2 mg/L 2,4-D promoted callus induction after 70 days of culture. Sub-culturing on the same medium enhanced the production of friable callus. Culture media containing higher concentrations of agar promoted the development of green somatic embryos from the callus. Respond of somatic embryogenesis was most successful with MS medium in 6.0 g/L agar supplemented with 5 mg/L BAP and 0.2 mg/L 2,4-D whereby the callus developed into green somatic embryos with an efficiency of 53%. This culture medium also produced the largest number plantlets.

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.

Somatic Embryogenesis and Plant Regeneration from Leaflets of Fritillaria ussuriensis M.

A method for the production of somatic embryos and subsequent plant regeneration for Fritillaria ussuriensis M. is described. Whole leaflet cxplants, derived from plantlets grown in vitro, formed light yellowish embryogenic calli within one month of culture in the dark. Somatic embryogenesis was obtained after a 28 d incubation on MS induction medium supplemented with 2 mg / L 2,4-D 0.5 mg / L BA, 0.5 mg / L KT and 500 mg / L CH followed by transfer to a second N medium containing 0.5 mg / L KT and 100 mg / L CH. Somatic embryos were transferred to MS medium with 0.1mg/ L NAA and placed in the light for regeneration. After two weeks, mature somatic embryo developed into whole plantlet.

Advances in Somatic Embryogenesis Research of Horticultural Plants

Advances in horticulture plant biotechnologies provide new opportunities for researchers to study the field of vegetative propagation and genetic engineering. Developments of clonal propagation methods, especially somatic embryogenesis (SE), have numerous potential applications. This paper reviewed progress of research on SE in horticultural plants in last decade;analyzed plant regeneration having both direct and indirect SE from the characteristics of occurrence means, but mainly in an indirect way;and discussed the impact factors of SE, as well as reviewed the research in the practical applications of horticulture plants SE in the practice.

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.

Characterization of Pro-embryogenic Calli and Somatic Embryogenesis of Byrsonima intermedia A. Juss.

Byrsonima intermedia A. Juss. is a species from the Brazilian Cerrado that produces edible fruits and, in common with other species from the Byrsonima genus, has pharmacological potential. Previous attempts to propagate the species through conventional methods showed difficulties. Thus, the purpose of this work was to characterize pro-embryogenic masses of Byrsonima intermedia callus, aiming for their in vitro propagation through somatic embryogenesis. Leaf segments from in vitro germinated seedlings were employed as explants for callus production. The calli were then subcultured and exposed to dyes to fulfill their embryogenic potential. Digitalizations of the cytological preparations were made in order to measure the area that was stained by both Aceto-Carmine and Evans-Blue, using image tool software. Somatic embryos were induced after treatments with l-naphthaleneacetic acid （NAA）. The percentages of double-colored areas （by Aceto-Carmine and Evans-Blue） were calculated and the data were analyzed by using the Skott-Knott test （P ≤ 0.05） and, the embryogenic callus, as well as the formation of somatic embryos were analyzed by using the Krsuskal-Wallis rank test （P ≤0.05）. The results show that double coloration is effective at identifying cells showing embryogenic potential. Early callus subculture phases show a larger percentage ofembryogenic area （83%） Somatic embryos were induced by using high auxin level.

Effect of Different Hormone Combinations on Somatic Embryogenesis in Cotton Cultivar Xinluzao 33(Gossypium hirsutum L.)

[Objective] This study was conducted to evaluate the efficiency of somatic embryogenesis and plant regeneration for an upland cotton cultivar Xinluzao33 under the induction of different hormone combinations and thus to determine the optimal hormone combination. [Method] Calli of Xinluzao33(Gossypium hirsutum L.) were induced from seedling hypocotyl tissue by a range of DK and BK combinations. Embryogenic calli and embryos were induced on callus-inducing medium(CIM) without any hormones. Callus appearance and quality were compared to determine which medium was the optimal for callus induction. Embryogenesis ratio was calculated to determine which medium was the best for somatic embryogenesis and plant regeneration. [Result] Callus induction rate was 100% in all the 12 hormone combinations.The calli were yellow or kelly, and their texture was loose or soft under low concentrations of DK combinations, green or white, variably compact under high concentrations of DK combinations. The calli induced by BK combinations were kelly or green, covering creamy white substance. The best medium for callus induction was DK6(0.05 mg/L 2, 4-D and 0.10 mg/L KT). Embryogenic calli were successfully induced from all the combinations. The efficiency of embryogenic callus induction,embryogenesis, and plantlet regeneration were significantly different among the 12 combinations. The result showed that the embryogenesis ratio was the highest in BK3 combination(0.50 mg/L IBA and 0.50 mg/L KT), 72.86% of embryogenic calli differentiated into somatic embryos after being cultured on CIM for 80 d, and80.93% of the somatic embryos finally regenerated into plants on SEM(somatic embryo induction medium). [Conclusion] These results indicate that hormone combination BK3(0.50 mg/LIBA and 0.50 mg/L KT) was the best medium for somatic embryogenesis and plant regeneration from Xinluzao33.

Molecular identification of plants regenerated from somatic hybridization between rice and apomictic Panicum

We attempted to introduce apomictic gene(s)into rice via somatic hybridization by usingapomictic Panicum maximum Jacq.as thedonor of apomictic gene(s).Protoplasts of rice derived from suspen-sion cells were inactivated with indoacetamide(IOA)and protoplasts of Panicum maximum

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.

Regeneration of Plants from Embryogenic Cell Suspensions of cv.“Datil”(Musa AA):Morphological Evaluation of Plants in the Field

The full cycle from embryogenic callus(EC)induction to field evaluation of regenerated plants is reported for the first time for banana cv.“Datil”(Musa AA).Immature male flowers were used for EC induction on a modified M1 culture medium.The ideal type of EC was obtained in the presence of 4.5μM and 9.0μM 2,4-dichlorophenoxyacetic acid(2,4-D).Embryogenic cell suspensions were established in both concentrations of 2,4-D;however,only the suspensions from ideal callus(IC)formed with 4.5μM of 2,4-D were regenerated.A histological study revealed the formation of structurally different cell masses during the regeneration phase of cv.“Datil”.The embryos germination was characterized by the growth cellular aggregates,indicating the possible occurrence of secondary embryogenesis.Development and acclimatization of the plants were carried out in a normal manner as observed for other cultivars.Field evaluation of the plants’genetic stability was based on observation of morphological traits.In the vegetative growth phase,6.61%of the plants presented drooping leaves and deformed semi-limbs.These traits,however,did not affect further plant growth during flowering and harvest phases.The produced fruits were of good quality.And the present study indicates that this cultivar may be stable genetically.

Development of an Efficient Plant Regeneration System for Pelargonium×Citrosum Vanleenii

[Objective]As a mosquito-repelling ornamental plant,Pelargonium×Citrosum Vanleenii(P.× Citrosum Vanleenii)is hard to be acquired because of its hybrid background,the paper was to a new regeneration system of(P.× Citrosum Vanleenii).[Method] By studying the influence of plant growth regulators(PGRs)on explant type(leaves and petioles),the optimal combinations of PGRs to maximize SELSs(somatic embryo-like structure)and buds were established.[Result]0.2 mg/L NAA+1.0 mg/L BA was best for LS(leaves segments)and 0.2 mg/L NAA + 1.5 mg/L BAs was best for PS(petioles segments).Cultured plantlets were successfully acclimatized in soil where they grew normally without any morphological variation.Although both LS and PS were usable,the leaf was a better explant for induction of embryogenic calli,somatic embryo-like structures and buds.[Conclusion]This work offered a rapid and efficient system for plant regeneration of P.×Citrosum Vanleenii.

DIRECT SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM PROTOPLAST-DERIVED CELLS OF WHEAT (Triticum aestivum L.)

Protoplasts were isolated from embryogenic calli derived from immature embryos ofwheat (Triticum aestivum L. cv. Jinan 177). The protoplasts were cultured in NMB mediumsupplemented with 1mg/L 2,4- D and 500mg/l casein hydrolysate (CH). The regenerated cellsfrom protoplasts divided to form somatic embryos directly. The somatic embryos grown to1.5- 2 mm in size directly developed into complete plants on solid MB medium without hor-mones.

Plant Regeneration From Soybean (Glycine max L.) Protoplasts via Somatic Embryogenesis

Protoplasts isolated from immature cotyledons of cultivated soybean (Glycine max L.) were cultured by Gelrite bead method in ZSP medium with soybean root nodule products (asparagine, glutamine, allantoin, allantoic acid, etc.) as the main nitrogen resources. Embryogenic calli were initiated and transferred to somatic embryo differentiation medium. Somatic embryos were formed from embryogenic calli and converted to plants. The regenerated plants normally grew and flowered.