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     

Somatic Embryogenesis and Plantlet Formation of Picea wilsonii Mast. in Ditterent Conditions

Embryogenic callus (translucent callus) was produced from immature zygotic embryos of Picea wilsonii Mast. Subsequently somatic embryogenesis occurred on the brown callus. The somatic embryos could be stimulated to developinto plantlets on the medium without hormone. Young somatic embryos were produced from embryogenic callus in liquid suspension culture, in which suspensor was several or more than ten times the size of the somatic embryo. The somatic embryo showed very similar to zygotic embryos in micro-section and living material.     

Somatic embryogenesis from immature zygotic embryos of annatto (<Emphasis Type="Italic">Bixa orellana</Emphasis> L.)

Summary In order to establish a protocol for somatic embryogenesis of annatto, Bixa orellana L., seeds (70 d after anthesis) from field-grown orchards had their coats dissected off, and immature zygotic embryos were excised aseptically from immature seeds collected from field-grown trees and used as explants. Embryos were cultured onto MS medium supplemented with or without different combinations of plant growth regulators and activated charcoal. Direct somatic embryogenesis was induced on explants incubated either in Murashige and Skoog (MS), 2,4-dichlorophenoxyacetic acid (2,4-D), and/or kinetin-supplemented media after 25 d of culture. The highest frequencies of embryogenesis and embryos per explant were obtained on medium containing 2.26 μM 2.4-D, 4.52μM kinetin, and 1.0 gl⁻¹ activated charcoal. The presence of charcoal was critical in increasing embryos per explant, to reduce the time to obtain somatic embryos, and mainly to prevent callus proliferation and subsequent indirect somatic embryogenesis. No embryogenic response was achieved when mature embryos were used. It was also observed that embryogenic response was significantly affected by genotype. Histological investigations revealed that primary direct somatic embryos differentiated exclusively from the protodermis or together with the outer ground meristem cell layers of the zygotic embryo axis, and from the protodermis of zygotic cotyledons. Diverse morphological differences, including malformed embryos, were observed among somatic embryos. In spite of the high frequencies of histodifferentiation of all embryo stages, a very low conversion frequency to normal plants from somatic embryos was observed.     

Somatic embryogenesis in pearl millet (Pennisetum glaucum): Strategies to reduce genotype limitation and to maintain long-term totipotency

Three genotypes of Pearl millet were screened in vitro for induction of embryogenic callus, somatic embryogenesis and regeneration. Shoot apices excised from in vitro germinated seedlings or immature embryos isolated from green house established plants were used as primary explants. The frequency of embryogenic callus initiation was significantly higher in shoot apices in comparison with immature zygotic embryos. Moreover, differences between genotypes were minimal when using shoot apices. Friable embryogenic calli (type II) developed on the initial nodular calli after 1 to 3 months of culture. The frequency of type II callus is related to the composition of the maintenance medium and they were more often found in ageing cultures. The transfer of embryogenic calli onto auxin-free medium was sufficient for inducing somatic embryo development in short-term culture (3 months) while a progressive loss in regeneration potential was observed with increasing time of subcultures. Maturation of embryogenic calli on medium supplemented with activated charcoal, followed by germination of somatic embryos on medium supplemented with gibberellic acid, restored regeneration in long-term cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

雷竹体细胞胚诱导的初步研究

以雷竹（Phyllostachys violascens）种胚为外植体, 脱分化产生愈伤组织, 愈伤组织诱导产生体细胞胚, 并发育成苗。实验结果表明： 愈伤组织诱导体细胞胚基本培养基为MS无机盐＋20 g·L^-1葡萄糖（glucose）＋10 mg·L^-1腺嘌呤（adenine sulfate）＋ 0.5 g·L^-1麦芽抽提物（malt extract）＋0.1 mg·L^-1 6-BA＋0.01 mg·L^-1氨氯吡啶酸（picloram）＋10 g·L^-1type A agar; 将基本培养基中的氨氯吡啶酸浓度升高至0.1 mg·L^-1即为愈伤组织诱导的最佳培养基, 早期子叶胚是愈伤组织诱导的最佳胚体; 愈伤组织在添加0.001 mg·L^-1TDZ和0.3 mg·L^-1ABA的愈伤组织最佳培养基上光照培养4周后, 去除其中的ABA, 并添加0.1 mg·L^-1NAA继续培养1个月, 体细胞胚数量最多可达87.43%, 该培养基是体细胞胚发生的最佳培养基; 将上述体细胞胚发生培养基中的6-BA浓度升高至1 mg·L^-1, 继代培养2个月后有小苗出现。组织学观察显示, 体细胞胚细胞核大、质浓且多数呈球形原胚状。     

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.     

Efficient plant regeneration of Malaysian <Emphasis Type="Italic">indica</Emphasis> rice MR 219 and 232 via somatic embryogenesis system

Improvement on rice plant regeneration system from an embryogenic callus using two Malaysian indica rice MR 219 and MR 232 was developed in this study. Different stages of rice explants (zygotic embryos) were tested for callus induction and regeneration using various carbon sources and amino acids. The present study shows that dough stage of zygotic embryos was the best stage of explants for the embryogenic callus induction and regeneration of both rice cultivars. The embryogenic callus nature with the compact structure was confirmed by scanning electron microscopy (SEM) analysis. Inclusion of maltose at the concentration between 10 and 20 mg/L shown higher responsive for the development of green somatic embryos and initiation of rice regeneration with an average of 29–37 (87–91%) regenerated plantlets, respectively. All in vitro regenerated rice plantlets were green, morphological and physiologically healthy condition. Rice plantlets were hardened in acclimatization room for 3 weeks and later transferred into soil with 95% survival in both varieties were recorded. Hence, regeneration system from zygotic rice embryos via somatic embryogenesis system was carried out successfully by using 10 g/L of maltose and combinations of glutamine, asparagine and arginine amino acids.     

杉木未成熟胚胚性愈伤组织诱导影响因素探析

该研究从基因型、6-BA浓度、外植体接种方式和合子胚发育阶段等方面,分析杉木未成熟胚胚性愈伤组织诱导的影响因素。结果表明:基因型、6-BA浓度、外植体接种方式和合子胚发育阶段均对胚性愈伤组织诱导频率有不同程度影响。6种基因型中,有3种基因型诱导出胚性愈伤组织,其中基因型S18胚性愈伤组织诱导频率最高,为11.7%。6-BA浓度在1.0~1.5 mg·L~(-1)范围内时,基因型S18的胚性组织诱导频率较高。以在去皮种子的一端切开一个小口的接种方式为最优,将合子胚剥出的方式易造成合子胚褐化死亡,将未剥皮的种子切开一个小口后直接接入培养基的方式不利于愈伤组织生成。适合胚性愈伤组织诱导的合子胚发育阶段为受精至胚器官分化阶段,合子胚进入成熟阶段后不利于胚性愈伤组织诱导,合子胚易生长成完整植株。     

Somatic embryogenesis of holm oak (<Emphasis Type="Italic">Quercus ilex</Emphasis> L.): ethylene production and polyamine content

The production of ethylene and the endogenous content of polyamines (PAs) have been recorded during the early development, maturation and germination of holm oak (Quercus ilex L.) somatic embryos. Ethylene production was high in embryogenic callus, immature somatic embryos and in explants showing secondary embryogenesis, while it was lower in mature and germinating somatic embryos. A higher ethylene production was also associated to the process of secondary embryogenesis. The exogenous application of 1-amino-1-cyclohexane carboxylic acid was not significantly effective on the production of ethylene by holm oak somatic embryos. Total PAs were more abundant in embryogenic callus and in both somatic and zygotic immature embryos, decreasing later on in the mature and germination phases. Immature somatic embryos of holm oak and immature zygotic embryos contain high levels of spermidine (Spd), which decreased during maturation and germination. Spermine (Spm) concentration was lower than that of Spd. Spm was more abundant in embryogenic callus and immature zygotic embryos than in mature embryos. Ethylene production did not seem to interfere with PA metabolism.     

栓皮栎体细胞胚胎发生的细胞组织学观察

以栓皮栎未成熟合子胚为外植体,在添加0.25mg/L 2,4-D和0.5mg/L 6-BA的MS培养基上6周可诱导产生2种类型的胚性愈伤组织,一种表面具光泽、白色;另一种表面光滑湿润具光泽,色泽淡黄或无色透明。组织切片表明,胚性愈伤组织的细胞体积小,细胞核大,细胞质浓,细胞排列紧密;非胚性愈伤组织细胞的体积大,细胞核小,细胞质稀薄。胚性细胞团培养在不含激素的培养基上可诱导产生体细胞胚。体细胞胚直接起源于胚性细胞团表皮或近表皮的单细胞,经历与合子胚相似的球形胚、心形胚、鱼雷胚和子叶胚发育阶段。所有发育时期的体细胞胚的胚轴、子叶均产生次生体胚,它们起源于细胞质较浓的表皮单细胞。     

Plant regeneration through somatic embryogenesis from callus induced on immature embryos of Alstroemeria spp. L.

Summary The plant regeneration ability of callus obtained from zygotic embryos of the monocot Alstroemeria spp. was studied. The best explants for somatic embryogenesis were immature zygotic embryos in half-ovules when the endosperm was still soft and white. For 2 genotypes embryogenic callus was induced on callus induction medium with a success rate of 54%. The best callus induction period was 10 weeks. The morphology of embryogenic callus was nodular. Somatic embryos were formed after transfer of the callus to regeneration medium. These somatic embryos revealed later on the typical features of zygotic Alstroemeria embryos. The total duration of the plant regeneration protocol, from inoculation till rooted plantlets ready for transfer to the greenhouse, was 28 weeks.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid     

Distribution and changes of reserve materials in cotyledon cells of Panax ginseng related to direct somatic embryogenesis and germination

Cotyledon explants from zygotic embryos of Panax ginseng produced somatic embryos on Murashige and Skoog basal medium without growth regulators. Somatic embryos developed directly from epidermal cells at the cotyledon base. Somatic embryos were always formed from the side of the cotyledon opposite to the one attached to the medium surface regardless of cotyledon orientation. The frequency of somatic embryo formation from the abaxial epidermis (66%) was much higher than that from the adaxial epidermis (12%). Differences in embryogenic response were likely related to cell structure. Abaxial epidermal cells were filled with reserve materials (lipid bodies), while adaxial epidermal cells were devoid of any prominent reserves. During germination, the reserve materials in the cells of the cotyledons disappeared rapidly. At the same time, the competency of somatic embryo formation from cotyledon explants declined rapidly to zero. Upon culture of the cotyledon explants (for somatic embryo induction), lipid bodies slowly disappeared, but starch grains accumulated prominently. Reserve materials disappeared after commencement of embryogenic cell division. During germination, lipid bodies rapidly disappeared, and chloroplasts developed instead of starch grains. Received: 29 January 1997 / Revised version received: 16 April 1997 / Accepted: 9 May 1997     

Induction of somatic embryogenesis in Pinus roxburghii Sarg.

Embryogenic calli were initiated from embryonic explants of Pinus roxburghii using female gametophytes containing immature pre-cotyledonary embryos. Zygotic embryos were collected at different developmental stages and cultured on various media. Initiation of embryogenic calli was achieved in pre-cotyledonary zygotic embryos of a 0.1-mm to 1.2-mm embryonal head on Douglus fir cotyledon revised medium (DCR) medium supplemented with 2,4-D or NAA and BA. Embryogenic callus development was initiated from the suspensor region of immature embryos. The method of immature embryo culture was significant as rapid embryogenic callus development occurred in megagametophytes where the suspensor was stretched onto the medium from the cut micropylar end. Sixty embryogenic lines were established from 2500 explants cultured during one season. A pro-embryo with six to eight meristematic cells and suspensor of six to ten long, vacuolated cells dominated the early phase of the callus development. Cleavage polyembryony occurred in proliferating callus, constituting a method of multiplication of these somatic embryos. Somatic embryos developed to stage-I and stage-II embryos on DCR medium supplemented with 5 μM 2,4-D or 10 μM NAA. Received: 30 June 1999 / Revision received: 15 November 1999 / Accepted: 3 December 1999     

Gene Expression Patterns During Somatic Embryo Development and Germination in Maize Hi II Callus Cultures

Gene expression patterns were profiled during somatic embryogenesis in a regeneration-proficient maize hybrid line, Hi II, in an effort to identify genes that might be used as developmental markers or targets to optimize regeneration steps for recovering maize plants from tissue culture. Gene expression profiles were generated from embryogenic calli induced to undergo embryo maturation and germination. Over 1,000 genes in the 12,060 element arrays showed significant time variation during somatic embryo development. A substantial number of genes were downregulated during embryo maturation, largely histone and ribosomal protein genes, which may result from a slowdown in cell proliferation and growth during embryo maturation. The expression of these genes dramatically recovered at germination. Other genes up-regulated during embryo maturation included genes encoding hydrolytic enzymes (nucleases, glucosidases and proteases) and a few storage genes (an α-zein and caleosin), which are good candidates for developmental marker genes. Germination is accompanied by the up-regulation of a number of stress response and membrane transporter genes, and, as expected, greening is associated with the up-regulation of many genes encoding photosynthetic and chloroplast components. Thus, some, but not all genes typically associated with zygotic embryogenesis are significantly up or down-regulated during somatic embryogenesis in Hi II maize line regeneration. Although many genes varied in expression throughout somatic embryo development in this study, no statistically significant gene expression changes were detected between total embryogenic callus and callus enriched for transition stage somatic embryos.Supplementary material is available for this article at     

Somatic embryogenesis in soybean via somatic embryo cycling

Summary The objectives of the present research were: a) to develop an efficient soybean embryogenic regeneration system characterized by a high frequency of explant response and a large number of somatic embryos per explant; b) to evaluate the factors affecting somatic embryogenesis via somatic embryo cycling; and c) to identify the origin of somatic embryos in the system. A highly improved and efficient system for soybean somatic embryogenesis was established using somatic embryo cotyledons and somatic embryo hypocotyl/radicle explants plated on α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented MS basal media. The system included somatic embryo cycling between liquid and solid medium and it consistently gave rise to a much higher frequency of explant response and a larger number of embryos per responding explant than those obtained from zygotic cotyledon explant tissues. Genotype, differences were observed for response in some of the treatments with cv “Fayette” being more responsive than “J103”. Histological studies revealed that somatic embryos induced in the somatic embryo cycling system originated almost exclusively from epidermal cells on both 2,4-D and NAA inductive media. The cells of the epidermis proliferated to produce somatic embryos directly without an intervening callus phase. A single-cell origin of somatic embryos was observed in cultures on a 40 mg/liter 2,4-D treatment. A large number of responding cells in the epidermis was also observed in the 10 mg/liter NAA treatment. The single-cell origin of somatic embryos from epidermal layers of the explant tissues should facilitate development of an efficient transformation system for soybean.     

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     

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.     

盐肤木体细胞胚胎发生及植株再生

以盐肤木(Rhus chinensis Mill.)幼胚为外植体,研究不同植物生长调节剂组合对其愈伤组织诱导及体细胞胚胎发生的影响,以建立盐肤木体细胞胚胎发生及植株再生体系。结果表明,最适愈伤组织诱导培养基为MS+6-BA 0.2 mg/L+2,4-D 1.0 mg/L,诱导率为84.57%,诱导出的初代愈伤组织白色或淡黄色,质地疏松,表面光滑,为非胚性愈伤。初代愈伤组织转移到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L培养基上培养1个月后,长出淡黄色质地紧密的胚性愈伤组织,诱导率高达100%,在此培养基上胚性愈伤组织增殖倍数为854.73%。所获得的胚性愈伤组织转接到1/2 MS+6-BA 2 mg/L+NAA 0.5 mg/L+蔗糖4%的培养基上培养1个月后可诱导体细胞胚胎发生,诱导率可达32.67%。诱导得到的体细胞胚胎经历球形胚、心形胚、鱼雷胚、子叶胚进一步分化发育成苗。无菌苗炼苗后栽种到泥炭土∶蛭石∶珍珠岩为2∶1∶1的生长基质上,能100%稳定成活。经过细胞学观察分析,体细胞胚的发育与合子胚相似。     

Improved somatic embryogenesis from Cocos nucifera (L.) plumule explants

Summary Coconut is one of the most recalcitrant species to regenerate in vitro. Although previous research efforts using plumule explants have resulted in reproducible somatic embryogenesis, efficiency is only 4 or 10 somatic embryos per plumule without or with a brassinolide treatment, respectively. In order to increase the efficiency of somatic embryogenesis in coconut, two different approaches were evaluated and reported here: secondary somatic embryogenesis and multiplication of embryogenic callus. Primary somatic embryos obtained from plumule explants were used as explants and formed both embryogenic callus and secondary somatic embryos. The embrogenic calluses obtained after three multiplication cycles were capable of producing somatic embryos. The efficiency of the system was evaluated in a stepwise process beginning with an initial step for inducing primary somatic embryogenesis followed by three steps for inducing secondary somatic embryogenesis followed by three steps for embryogenenis callus multiplication, and finally production of somatic embryos from callus. The total calculated yield from one plumule was 98 000 somatic embryos. Comparing this to the yield obtained from primary somatic embryogenesis results in about a 50 000-fold increase. When compared to the yield previously reported in the literature with the use of a brassinolide treatment, it is about a 10 000-fold increase in yield. The present protocol represents important progress in improvement in the efficiency of coconut somatic embryo production.     

In vitro somatic embryogenesis in two major rattan species: Calamus merrillii and Calamus subinermis

Summary Occurrence of somatic embryogenesis in in vitro cultures of Calamus merrillii and Calamus subinermis, two major largecaned rattan species, was scientifically demonstrated for the first time. Tissue responsiveness varied markedly according to the species and the type of primary explants used when initiated on 10.4–31.2 μM picloram-enriched Murashige and Skoog callus induction media. In C. merrillii, within 6 wk after inoculation, 84% of the leaf and 90% of the zygotic embryo explants produced friable embryogenic calluses, by contrast with those formed by 74% of the root explants. In C. subinermis, callogenesis was observed only 6 mo. after inoculation in 68% of root and 48% of zygotic explants. Leaf explants did not respond at all. Only root-derived calluses developed into nodular embryogenic structures. Irrespective of these initial differences, the further steps of the somatic embryogenesis developmental pattern was similar for both species. Histological analyses established that callus formation took place in the perivascular zones, and could give rise to embryogenic isolated cells from which the proembryos were derived. Reducing the picloram concentration stimulated the maturation process resulting ultimately in the germination of somatic embryos that exhibited bipolar development, despite an apparent lack of starch and protein reserves. The somatic embryo-derived plantlets of C. merrillii, overall more prone to somatic embryogenesis than C. subinermis in the given conditions, were successfully acclimatized to outdoor conditions.