Somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Terminalia chebula</Emphasis> Retz.: an important medicinal tree

Somatic embryogenesis was obtained from cotyledon and mature zygotic embryo callus cultures of Terminalia chebula Retz. Callus cultures of cotyledon and mature zygotic embryo were initiated on induction medium containing Murashige and Skoog (MS) nutrients with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) either 0.01 or 0.1 mg/l Kinetin and 30 g/l sucrose. Induction of somatic embryogenesis, proliferation and development was obtained through different culture passages. Embryogenic cotyledon callus with globular somatic embryos was obtained on MS basal medium supplemented with 50 g/l sucrose. Globular somatic embryos were observed from mature zygotic embryo callus on induction medium. Different stages of somatic embryo development from cotyledon and mature zygotic embryo calluses were observed on MS basal medium supplemented with 50 g/l sucrose after 4 weeks of culture. Histological studies have revealed the developmental stages of somatic embryos. A maximum of 40.3±1.45 cotyledonary somatic embryos/callus was obtained from mature zygotic embryo compared to 7.70±0.37 cotyledonary somatic embryos/callus initiated from cotyledons. Germination of somatic embryos and conversion to plants were achieved. Highest frequency of germination (46.66±0.88) of somatic embryos was obtained on MS basal medium containing benzyladenine (0.5 mg/l) with 30 g/l sucrose.     

Single-cell origin and development of somatic embryos in Picea abies (L.) Karst. (Norway spruce) and P. glauca (Moench) Voss (white spruce)

The origin and development of somatic embryos in calli initiated from immature zygotic embryos of Picea abies (L.) Karst. (Norway spruce) and P. glauca (Moench) Voss (white spruce) was studied. Immature zygotic embryos cultured on callus induction medium produced two types of white calli that were phenotypically different from one another. The callus that proliferated from the hypocotyl region was white to translucent, glossy, mucilaginous and embryogenic. The callus mass which originated from the radicle end was reddish-white, nonmucilaginous and nonembryogenic. Whole mount preparations of the entire explant with two different types of calli showed the presence of embryogenic cells in the mucilaginous callus mass derived from the hypocotyl region of the zygotic embryo. The origin of somatic embryos in both Norway and white spruce could be traced to single cells of the hypocotyl callus.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine     

Regeneration from intact and sectioned immature embryos of barley (Hordeum vulgare L.): the scutellum exhibits an apico-basal gradient of embryogenic capacity

Immature zygotic embryos from spring barley cv. Dissa were used to induce somatic embryogenenesis. Up to 158 germinated somatic embryos could be recovered per plated zygotic embryo. Critical factors for obtaining a high yield of regenerants were the size of the explant, the level of 2,4-D used for callus induction and the careful division of callus at each subculture. Use of microsections of immature embryos as explants revealed a pronounced gradient of callus formation and embryogenic response across the scutellum. Sections from the scutellar tissue at the coleoptilar end of the embryo gave the most callus and were highly embryogenic. The regeneration response of sectioned explants was comparable to that recovered from intact embryos of similar size.     

黄山栾树体细胞胚的发生和组织学观察

黄山栾树无菌苗的节间和叶柄离体培养后,其体细胞胚发生的结果表明：节间愈伤组织可诱导产生体细胞胚,而叶柄愈伤组织则生根：节间愈伤组织诱导培养基为MS＋3．0mg．L～2,4．D＋0．5～3．0mg．L-1NAA;节间胚性愈伤组织诱导培养基为MS＋2．0nag．L-2,4-D;胚性愈伤组织转移到无植物生长调节剂的MS培养基上可发育成正常植株。组织学观察表明,体细胞胚在胚性愈伤组织中有的发生于愈伤组织表层细胞,有的发生在愈伤组织内部。黄山栾树体细胞胚的形成经历球形胚、心形胚、鱼雷胚和子叶胚几个阶段,这与合子胚的发育途径相似。     

Somatic Embryogenesis in Capparis decidua (Forsk) Edgew — A Multipurpose Agroforestry Plant

An efficient protocol for direct somatic embryogenesis in Capparis decidua has been developed. Mature zygotic embryos cultured in Murashige and Skoog (MS) liquid medium with 2,4-0 (0.1 mg l^?1) and BA (0.5 mg l^?1) produced somatic embryos directly without an intervening callus phase from the subepidermal cells. Treatment with ABA promoted maturation of somatic embryos and BA (1 mg l^?1)promoted germination. One zygotic embryo produced approximately 230 somatic embryos within 14 to 15 weeks. Embryos germinated in eight weeks and acclimatized plants were transferred to pots.     

Development of a cyclic somatic embryogenesis regeneration system for leek (Allium ampeloprasum L.) using zygotic embryos

Summary In leek (Allium ampeloprasum L.) a cyclic system of somatic embryogenesis was developed. Somatic embryos used for cyclic embryogenesis were able to develop the same type of embryogenic callus as zygotic embryos in the primary cycle. For the first time a comparison of the efficiencies of both expiants was made. Ten families were investigated for somatic embryogenesis. There was a genetic relationship with respect to somatic embryo production between the reciprocal crosses. From each family one genotype was selected for investigating cyclic somatic embryogenesis. Different levels of somatic embryo production were found between the expiants of zygotic and somatic embryos. The two best genotypes, 92.001-03 and 92.002-33 produced twice as many somatic embryos as the overall average. On average, 56% of the somatic embryos finally developed into greenhouse plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - MS medium Murashige and skoog (1962) basal medium     

Morphology and Anatomy of Pisum Sativum Somatic Embryos

The morphological and anatomical aspects of direct and indirect somatic embryogenesis in pea were described. Direct embryos were induced from shoot apical meristems of 3 to 5-d-old pea seedlings, embryogenic callus originated from immature pea zygotic embryos or shoot apices. Auxin (picloram, 2,4-dichlorophenoxyacetic acid) was necessary to induce somatic embryos. The developmental stages typical for pea zygotic embryos were detected. Globular and heartshaped somatic embryos were morphologically similar to their zygotic counterparts; in contrast, torpedo and cotyledonary somatic embryos displayed great morphological variation, which affected mainly cotyledons (size, shape, number). Based on anatomical sections, possible ways of somatic embryo formation and localization of initiation sites within primary explant tissue have been proposed. The multicellular origin of somatic embryos is supposed in both systems of pea somatic embryogenesis under investigation.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Effect of plumule and radicle on somatic embryogenesis in the cultures of ginseng zygotic embryos

Immature zygotic embryos of ginseng produced somatic embryos on MS medium without growth regulators. However, in the culture of mature zygotic embryos, excision of the embryo was required for somatic embryo induction. Somatic embryos formed only on excised cotyledons without an embryo axis or on excised embryos without the plumule and radicle of the axis. This observation suggests that the axis tip of the embryo might suppress somatic embryo production although the cotyledon tissues have predetermined embryogenic competency. To clarify the role of the embryo axis on somatic embryo formation, excised plumules or radicles were placed in direct contact with the basal cut-ends of cotyledons. The adhesion of plumules or radicles highly suppressed somatic embryo formation from cotyledon explants. When an agar block containing exudate from excised plumules or radicles was placed in contact with the cut end of the cotyledon, a similar inhibition was observed. These results suggest that embryogenic competence is suppressed by endogenous inhibitors present in the axis tip of the zygotic embryo.     

Role of 2,4-dichlorophenoxyacetic acid (2,4-D) in somatic embryogenesis on cultured zygotic embryos of Arabidopsis: cell expansion, cell cycling, and morphogenesis during continuous exposure of embryos to 2,4-D

The relationship between cell expansion and cell cycling during somatic embryogenesis was studied in cultured bent-cotyledon-stage zygotic embryos of a transgenic stock of Arabidopsis thaliana harboring a cyclin 1 At:β-glucuronidase (GUS) reporter gene construct. In embryos cultured in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), following a brief period of growth by cell expansion, divisions were initiated in the procambial cells facing the adaxial side at the base of the cotyledons. Cell division activity later spread to almost the entire length of the cotyledons to form a callus on which globular and heart-shaped embryos appeared in about 10 d after culture. Anatomical and morphogenetic changes observed in cultured embryos were correlated with patterns of cell cycling by histochemical detection of GUS-expressing cells. Although early-stage somatic embryos did not develop further during their continued growth in the auxin-containing medium, maturation of embryos ensued upon their transfer to an auxin-free medium. In a small number of cultured zygotic embryos the shoot apical meristem was found to differentiate a leaf, a green tubular structure, or a somatic embryo. Contrary to the results from previous investigations, which have assigned a major role for the shoot apical meristem and cells in the axils of cotyledons in the development of somatic embryos on cultured zygotic embryos of A. thaliana, the present work shows that somatic embryos originate almost exclusively on the callus formed on the cotyledons. Other observations such as the induction of somatic embryos on cultured cotyledons and the inability of the embryo axis (consisting of the root, hypocotyl, and shoot apical meristem without the cotyledons) to form somatic embryos, reaffirm the important role of the cotyledons in somatic embryogenesis in this plant.     

花楸合子胚诱导体细胞胚胎发生研究

分别以完整成熟胚、切去一个子叶的成熟胚和切下的子叶为外植体,以MS为基本诱导培养基、1/2MS为基本分化培养基,进行了花楸体细胞胚胎发生研究。结果表明:以完整合子胚作为外植体的体胚诱导率最高,为100%,最佳植物生长调节剂组合为5 mg.L-1NAA+2 mg.L-16-BA;NAA和6-BA浓度及二者的交互作用对愈伤组织和体胚诱导率的影响极显著;光照配合延长继代间隔时间有利于体胚发生。实体观察结果表明,花楸体胚发生方式有直接发生和间接发生两种;体胚发育经历了球形期、心形期、鱼雷形期和子叶期。组织学观察结果表明,体胚具有两极性,子叶期体胚结构完整。     

糜子离体体细胞胚胎发生的组织学研究

利用组织学连续石腊切片的研究方法,观察了糜子组织培养中植株再生的过程,从而证明了植株再生是通过体细胞胚胎发生途径的。结果表明:(1) 糜子成熟胚培养首先从下胚轴及胚根区愈伤组织化;(2) 体细胞胚起源于下胚轴及胚根维管束周围愈伤组织中单个离散的胚性细胞;(3) 糜子离体体细胞胚有与典型禾谷类作物合子胚大致相似的发育过程。     

SOMATIC EMBRYOGENESIS AND PLANT REGENERATION FROM IMMATURE ZYGOTIC EMBRYOS OF MUSCADINE GRAPE (VITIS ROTUNDIFOLIA) CULTIVARS

Embryogenic cell lines of Vitis rotundifolia were produced from immature zygotic embryo explants obtained by culturing ovules, harvested at 20 d postanthesis, for 8 wk and then dissecting embryos from them. Ovules cultured on Nitsch and Nitsch medium with naphthoxyacetic acid and benzyladenine (BA) produced a brown exudate, necessitating three transfers to fresh medium at 2-wk intervals during the 8-wk culture cycle. Zygotic embryos that were subsequently isolated from cultured ovules and placed on the same medium produced a heterogenous callus from which eventually emerged embryogenic cell lines. A higher percentage of ovules from cultivars ‘Dixie’, ‘Fry’, ‘Nesbitt’, and ‘Welder’ produced zygotic embryos (31%–39%) than did those from ‘Carlos’ (3%). A higher percentage of ‘Fry’ ovules produced embryogenic lines from cultured zygotic embryos (6.3%) than did those of the other four cultivars (1%–1.6%). Embryogenic cell lines were white and composed of variably sized cell clusters, somatic embryos, and embryonic tissue embedded in a watery matrix. These lines were maintained for over 1 yr on modified Murashige and Skoog (MS) medium lacking growth regulators by transfer of selected cell clusters every 6 wk. White, opaque somatic embryos grew directly from cell clusters and passed through recognizable developmental stages. Germination was induced by transfer of somatic embryos to MS medium with BA. Although 80%–100% of embryos germinated, plant recovery was low due to poor shoot development.     

Factors important for somatic embryogenesis in zygotic embryo explants ofCapsicum annuum L.

We used four cultivars ofCapsicum annuum L.—Sweet Banana, California Wonder, Yolo Wonder, and Ace—to reexamine the critical factors influencing somatic embryogenesis from zygotic embryo explants, as reported in the literature. When we followed the protocol of Buyukalaca and Mavituna (1996), which had induced somatic embryogenesis from mature zygotic embryos of cv. Ace, only callus was formed without embryogenesis from our mature zygotic embryo expiants. Using the procedures of Harini and Lakshmi Sita (1993) and Binzel et al. (1996), with some modifications, we were able to induce somatic embryogenesis in all four cultivars. Rates of conversion were significantly reduced, from 75% and 65% to 40% and 28% in ’Sweet Banana’ and ’California Wonder’, respectively, when the immature zygotic embryo expiants were held on the induction medium for longer than two weeks. Likewise, somatic embryogenesis of ’Yolo Wonder’ was not observed if the induction medium was supplemented with 10% glucose or fructose, or without 10% sucrose. For somatic embryo induction and eventual plantlet conversion in Yolo Wonder’, maltose could adequately replace sucrose. In all four cultivars, somatic embryos were initiated from immature zygotic explants on media with or without coconut water, under both light and dark conditions.     

Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy

Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.     

Starch deposition and amylase accumulation during somatic embryogenesis in bamboo (Dendrocalamus hamiltonii)

In monocots, the zygotic embryo is protected and nourished by an endosperm. In the present study starch deposition and amylase accumulation was noticed during somatic embryogenesis in stem callus of a bamboo, Dendrocalamus hamiltonii. SEM studies revealed that starch grains were clearly visible in the scutellum during the maturation stage of the somatic embryo. As the somatic embryo developed further, the scutellum got reduced with corresponding increase in amylase. The amylase activity was tested periodically at different developmental stages of embryos. The role of scutellum in somatic embryos for starch deposition and amylase accumulation is discussed.     

Developmental Aspects of Soybean (Glycine max) Somatic Embryogenesis

A detailed study of the developmental aspects for soybean somaticembryogenesis was undertaken with emphasis on biochemical andhistological markers. The various stages of somatic embryo developmentin callus cultures have been identified and characterized. Germinatingembryos could be converted to fertile plants at a high frequency(90%). Dicamba (3, 6-dichloro-o-anisic acid) was found to bethe auxin of choice for the clear distinction of the variousdevelopmental phases of soybean somatic embryos. Differencesin their protein patterns were determined using polyacrylamidegel electrophoresis. This analysis revealed distinguishabledifferences in protein profiles amongst the various developmentalstages, especially in heart stage embryos. Histological studieson somatic embryos revealed specific tissue types which closelyresemble those reported for zygotic embryos. Further evidenceis provided that there is a close similarity in tissue differentiation,between somatic and zygotic embryogenesis although there aresome unique features in the development of somatic embryos. Glycine max, callus cultures, developmental stage, liquid cultures, neomorphs, plant regeneration, stage specific proteins, histology     

Direct somatic embryogenesis and plant regeneration from immature embryos of hybrid sunflower (Helianthus annuus L.) on a high sucrose-containing medium

Somatic embryos of sunflower (Helianthus annuus) were obtained by placing immature zygotic embryos on a high sucrose (12%) containing medium. The somatic embryos were first observed 6 days after culture and a callus intermediate was not formed. Histological examination revealed the classical stages of embryo development. The somatic embryos proliferated directly from the surface of the zygotic embryos and germinated after placement on a low sucrose medium. Seeds have been obtained from regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - BAP 6-benzylamino purine. Salaries and research support were provided by State and Federal funds appropriated to OSU-OARDC. Journal Article No. 67–87     

Somatic embryogenesis to overcome low seed viability and conserve wild banana (Ensete superbum (Roxb.) Cheesman)

The seed viability, ex vitro germination, and percentage of in vitro zygotic embryo germination were found to be very low in Ensete superbum (Roxb.) Cheesman. Only 33.33% of seeds were viable, and the ex vitro germination percentage was only 5%, while the percentage of in vitro zygotic embryo germination was 33%. Somatic embryogenesis experiments produced competent callus on Murashige and Skoog (MS) medium supplemented with 2.5 mg L⁻¹ 2,4-D and 3 mg L⁻¹ BAP from inflorescence explants. The embryogenic callus produced the maximum number of somatic embryos on MS basal medium kept in a dark chamber for 15 wk. Half-strength MS medium supplemented with 500 mg L⁻¹ glutamine was optimal for somatic embryo germination and development of plantlets. Regenerated plants had 80 to 90% survival rate. Therefore, somatic embryogenesis can be considered as an efficient method to overcome a drastic reduction in population and to achieve germplasm conservation.

     

The effect of auxin type and concentration on pecan (Carya illinoinensis) somatic embryo morphology and subsequent conversion into plants

Summary Embryogenic cultures were initiated from immature pecan zygotic embryos. Explants were induced for one week on Woody Plant Medium with either -naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid at 2, 6 or 12 mg/l, then subcultured monthly to fresh basal medium. Observations were made on callus production, embryo formation, and embryo morphology. Somatic embryo morphology and overall callus proliferation were affected by auxin type. Callus proliferation was less extensive and more somatic embryos resembling zygotic embryos were obtained from cultures initiated with -naphthaleneacetic acid than with 2,4-dichlorophenoxyacetic acid. Repetitive somatic embryogenesis was obtained in all auxin treatments. Conversion into plantlets was affected by somatic embryo morphology in that embryos with poorly developed apices exhibited lower percentages of conversion than those with well developed single or multiple apices. Consequently, although more embryos were obtained with 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid was the superior auxin for production of somatic embryos more likely to convert into plants.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - WPM Woody Plant Medium (Lloyd & McCown 1980)