Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba×Abies cephalonica,and Abies alba×Abies numidica)

Summary Embryogenic cultures were established from silver fir (Abies alba Mill.) female megagametliophytes with developing embryos and from excised mature embryos after pollination with Abies cephalonica Lond. or Abies numidica DeLann pollea The frequency of embryogenic callus formation was dependent on genotype, collection time, medium and explants used. The embryogenic callus initiation potential of megagamethophytes with developing embryos in both hybrids was higher in early July and dropped as the zygotic embryos matured. Excised cotyledonary embryos were less suitable for induction of embryogenic cultures. SH medium supplemented with 1mg/l BAP was the most efficient for callus induction and maintenance. Cultures were composed of early somatic embryos with an embryonal mass formed of highly cytoplasmic cells, rich in cell organelles and a suspensor built up by vacuolated, strongly elongated cells. Maturation of embryos was detected with the formation of bipolar structures with shoot and root apices. Nutrition reserves were observed in cells of embryos cultured on DCR medium containing 1 or 10 mg/l ABA. Cotyledon formation, hypocotyl elongation and low frequency germination occured following transfer of the embryos to the same medium without ABA.     

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.     

Somatic embryogenesis and plant regeneration from protoplasts isolated from embryogenic cell suspensions of wheat (Triticum aestivum L.)

We describe the early formation of somatic embryos followed by plant regeneration from protoplasts isolated from an embryogenic wheat cell suspension, which was initiated from small granular (0.2 to 1 mm in size) embryogenic calli. These granular calli formed embryogenic cell suspensions within 20 days in liquid culture, and were selected gradually from young inflorescence-derived nodular embryogenic calli of the winter wheat cv. Kehong 1041. The division frequency of protoplasts was 11 to 16%, and the frequency of differentiation into plants was about 0.001% (number of plants formed divided by the total number of protoplasts plated). About 20% of somatic embryos present in the culture formed directly from protoplast-derived cells within 15 days of cultures.     

Embryogenesis and plant regeneration of Medicago spp. in tissue culture

Ten cultivars and breeding lines from two species of alfalfa (Medicago media and M. sativa) were screened for their ability to produce embryos and plantlets from the root and hypocotyl under three different tissue culture protocols. The three protocols differed in basal salt composition, vitamins, hormones and cytokinin additions. That protocol having a high 2–4,D low cytokinin induction step gave the highest percentage of embryogenic calli in some cultivars and lines. M. media cultivars and breeding lines had a high percentage of embryoid formation. M. sativa cultivars gave no embryoid formation. Two M. media breeding lines (Br1 and Le1), which were intermediate in the percentage of embryogenic calli formed from explants, had the highest number of regenerated plants established in soil. The creeping rooted M. media cultivar Heinrichs produced the highest percentage of embryogenic calli from explants but most of these embryoids were abnormal and failed to grow in soil or vermiculite. Accordingly, successful regeneration is directly related to the quality and quantity of the embryoids produced. Respectively: Biotechnology Department, Alberta Research Council, Agriculture Canada, Beaverlodge, Alberta, and University of Alberta, Edmonton, Alberta, Canada     

Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.)

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.Abbreviations NAA 1 naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog - BA 6 benzylamino purine - 2i P N⁶-(²-isopentenyladenine     

Acquisition of embryogenic potential in carrot cell-suspension cultures

Embryogenic suspension cultures of domesticated carrot (Daucus carota L.) are characterized by the presence of proembryogenic masses (PEMs) from which somatic embryos develop under conditions of low cell density in the absence of phytohormones. A culture system, referred to as starting cultures, was developed that allowed analysis of the emergence of PEMs in newly initiated hypocotyl-derived suspension cultures. Embryogenic potential, reflected by the number of FEMs present, slowly increased in starting cultures over a period of six weeks. Addition of excreted, high-molecular-weight, heat-labile cell factors from an established embryogenic culture considerably accelerated the acquisition of embryogenic potential in starting cultures. Analysis of [³⁵S]methionine-labeled proteins excreted into the medium revealed distinct changes concomitant with the acquisition of embryogenic potential in these cultures. Analysis of the pattern of gene expression by in-vitro translation of total cellular mRNA from starting cultures with different embryogenic potential and subsequent separation of the [³⁵S]methionine-labeled products by two-dimensional polyacrylamide gel electrophoresis demonstrated a small number of abundant in-vitro-translation products to be present in somatic embryos and in embryogenic cells but absent in nonembryogenic cells. Several other in-vitro-translation products were present in explants, non-embryogenic and embryogenic cells but were absent in somatic embryos. Hybridization of an embryoregulated complementary-DNA sequence, Dc3, to RNA extracted from starting cultures showed that the corresponding gene is expressed in somatic embryos and PEMs but not in non-embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - cDNA complementary DNA - PAGE polyacrylamide gel electrophoresis - PEM proembryogenic mass     

Plant regeneration through somatic embryogenesis from mature seed and young inflorescence of wild rice (Oryza perennis Moench)

Somatic embryogenesis in the wild rice species (Oryza perennis) was induced from cultured mature seeds and young inflorescences. Murashige and Skoog's (MS) medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l BAP was used for induction of a compact, white nodular callus and somatic embryos. Plant regeneration occurred with the tranfer of the nodular callus to MS basal medium containing 0.5 mg/l IAA, 0.5 mg/l NAA, 4 mg/l BAP and 500 mg/l casein hydrolysate. The embryogenic nature of the callus from both explants was maintained over 10 subcultures for about 12 months. Plant regeneration with respect to the number of calli plated from the 6th to 10th passage varied from 80% to 60% for young inflorescence derived callus and from 75% to 69.8% for seed-derived callus.Abbreviations MS Murashige and Skoog medium - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthalene acetic acid - CH casein hydrolysate     

Screening of diploid Medicago sativa germplasm for somatic embryogenesis

Nineteen accessions of diploid Medicago sativa L. belonging to the four subspecies sativa, caerula, falcata and xvaria were screened for their ability to produce somatic embryos on hypocotyl-derived callus. Two medium protocols were used in this study, a three-step sequence with exposure of the callus cultures to a high 2,4-D concentration and a two-step sequence without exposure to a high 2,4-D concentration. Considerable variation for callus proliferation was observed. In general, the diploid M. sativa accessions showed poor regenerability and it was not possible to correlate high regeneration frequencies with a particular germplasm source. It was, however, possible to identify regenerable genotypes in all four subspecies. One falcata accession produced somatic embryos on the callus induction media at high frequencies. This response was also obtained with a few genotypes from one xvaria accession. All regenerable plants were maintained as shoot cultures and were able to form somatic embryos on petiole-derived calli.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2iP iso-pentyladenine - NAA -naphthaleneacetic acid Contribution No. 772 Ottawa Research Station     

The somatic embryogenesis and establishment of transformation experiment system in Larix principis-Rupprechtii]

Larix principis-Rupprechtii is one of the superior afforestation forest trees growing in north China. Embryogenic cultures were initiated from immature zygotic embryos of Larix principis-Rupprechtii on S culture medium containing 2, 4-D 0-2.2 mg/L, KT and BA each at 0-0. 8 mg/L. Embryogenic calli were subcultured and multiplicated on S + B culture medium containing dropping off each hormone concentration. We set up 33 steady-going embryogenic cell lines; We studied on the growth stage and genotype differences of every embryogenic cell lines; and Finded more than 10 high-frequency somatic embryogenesis cell lines such as 2K, 2T, 2I, 2J, 3C etc.. The number of 2T somatic embryos reaches 314/per gram of embryogenic tissue and the number of 3C somatic embryos is 185/per gram of embryogenic tissue. The re-induction method of Larix principis-Rupprechtii from somatic embryos was used to produce renewable embryogenic cultures and steady-going embryogenic cell lines effectively. Mature somatic embryos can germinate and develop further into plantlets when they are isolated and cultured on a hormone-free WPM culture medium. The regeneration plantlets were obtained. Furthermore, the transformation with a truncated gene of Bacillus thuringensis (B. t) were carried out, the PCR showed positive results, because of this, embryogenic cell line of Larix principis-Rupprechtii can be used for transformation experiments to support further breeding in forestry.     

Somatic embryogenesis and plant regeneration through cell suspension in diploid (AB) banana cultivar Elakki Bale (syn Neypoovan)

An efficient protocol was developed using cell suspensions for somatic embryogenesis and plantlet regeneration in a most popular diploid AB banana (M.accuminata X M.bulbisiana hybrid) cv. Elakki Bale (syn Neypoovan) known for its taste and keeping quality in southern India. Floral primodia from position 8–16 of male inflorescence which were more responsive for embryogenesis were used as explants for the embryogenic callus production in MS media supplemented with different concentration of 2,4-D. A concentration of 18.1 μM 2, 4-D produced maximum embryogenic calli in 1 % of the explants inoculated. Embryogenic calli on repeated sub culturing on MA2 media produced good embryogenic cell suspensions (ECS). Microscopic examination of ECS showed globular, smaller with dense cytoplasm filled with starchy granules characteristic of embryogenic cells. Highest number of somatic embryos (189) was produced on modified MA3 media. A germination percentage of 31 % were observed in BAP 22.19 μM concentration. Regenerated plants with normal shoot and root were hardened in soilrite. Direct somatic embryogenesis and plant regeneration was also noticed in embryogenic calli which did not pass through the ECS stage. The protocol optimized for somatic embryogenesis through cell suspension and also direct embryogenesis leading to plantlet regeneration can be used for the micropropagation and genetic manipulation.     

华北落叶松体细胞胚胎发生及遗传转化实验系统的建立(简报)

华北落叶松(Larix principis-Rupprechtii)是我国北方中高山地区重要的针叶速生用材树种,进行其体细胞胚胎发生和植株再生的研究,在针叶树无性快速繁殖及基因工程育种上有其特殊的用途,既可为针叶树无性系林业提供产业化途径,也可作为目的基因遗传转化实验系统。针叶树的基因转化相对较难,再生更属不易,Lelu等报道过杂种落叶松与欧洲落叶松体细胞胚胎发生方面的研究;而我国尚未见有落叶松体细胞胚胎发生的研究报道。我们     

Somatic embryogenesis in peach palm using the thin cell layer technique: induction, morpho-histological aspects and AFLP analysis of somaclonal variation

BACKGROUND AND AIMS: The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. METHODS: TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0-600 microM Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. KEY RESULTS: Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150-600 microM Picloram (83-97%, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43% embryogenic callus production from shoot meristem TCL on 300 microM Picloram. In maturation conditions, 34+/-4 somatic embryos per embryogenic callus were obtained, and 45.0+/-3.4% of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80% survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92% of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. CONCLUSIONS: The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees.     

Electric field mediated stable transformation of carrot protoplasts with naked DNA

We have developed an electroporation procedure for the transformation of carrot protoplasts with Ti-plasmid DNA from Agrobacterium tumefaciens. The uptake of pTiC58 into carrot protoplasts was mediated by high voltage electrical pulses at field strengths from 0.5 to 3.8 kV/cm. Protoplast regeneration, somatic embryogenesis and plantlet regeneration were unaffected by the electroporation conditions selected for DNA uptake. Uptake of plasmid pTiC58 resulted in hormone independent regeneration of carrot protoplasts. Transformed somatic embryos were detected in carrot cultures 45 days after electroporation. The transformed somatic embryos developed into teratomas which synthesized nopaline. Hybridization was obtained between a labeled T-DNA fragment from pTiC58 and DNA fragments from 4 month old teratomas regenerated from electro-transformed protoplasts. Based on the number of somatic embryos regenerated after electro-transformation, a frequency of 1.6×10² transformants/10⁴ somatic embryos/g pTiC58 DNA was obtained.Abbreviations PEG polyethylene glycol - 2,4-D 2,4-dichlorophenoxyacetic acid - MES morpholinoethane sulfonic acid - DMSO dimethyl sulfoxide - HSV Herpes Simplex virus - TK thymidine kinase     

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     

Plant regeneration through somatic embryogenesis from suspension cultures of a minor millet,Paspalum scrobiculatum

Compact, friable and embryogenic calli were initiated from immature inflorescences and young leaf bases of one week old seedlings of Paspalum scrobiculatum cultured on MS medium supplemented with 2,4-D. A stable, embryogenic suspension culture was initiated from these calli and maintained in a liquid version of the same MS medium. Embryogenic calli and somatic embryos were obtained by plating suspension culture cells onto semi-solid medium containing 2,4-D. Complete, normal plantlets developed on 2,4-D free medium at a high frequency from somatic embryos. NAA and BAP in the medium promoted plant development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - MS Murashige and Skoog (1962) - CM Coconut milk     

Development of an efficient plant regeneration system for Russian wildrye (<Emphasis Type="Italic">Psathyrostachys juncea</Emphasis>)

Russian wildrye [Psathyrostachys juncea (Fisch.) Nevski] is a cool-season forage grass with a broad adaptation to semi-arid regions of North America. In order to explore the potential of biotechnology for genetic improvement of this important forage species, we developed an efficient tissue culture system. Embryogenic calli were induced from mature embryos with an induction frequency in the range of 2-7%. The selected highly embryogenic calli allowed the regeneration of dozens of plants from a single callus. Individual embryogenic calli were then used to establish single genotype-derived suspension cultures. Eighteen embryogenic cell suspension lines were established from three cultivars (Bozoisky-Select, Sawki and Tetracan). A relatively high green plant regeneration frequency, up to 70%, was achieved from plated cell clusters of the established suspension cultures. The regenerated plants were fertile after two winters of vernalization in the field. This efficient plant regeneration system provides a solid basis for generating transgenic plants.     

Long-term optimized embryogenic cultures in durum wheat (Triticum durum Desf.)

Five varieties of durum wheat: Appulo, Ofanto, Latino, Creso, and Castello (Triticum durum Desf.) adapted to the semi-arid mediterranean environment have been tested for their in vitro response. Compact, embryogenic, highly regenerable calli originated from primary callus derived through proliferation of the scutellum of immature embryos explanted in the presence of 2,4 dichlorophenoxyacetic acid. Selective subculture of the white, compact, embryogenic sectors led to the establishment of long-term cultures. Regeneration occurred on hormone-free medium either via germination of somatic embryos, or via multiple-shoot formation probably due to precocious germination of somatic embryos. The three varieties, Ofanto, Creso and Appulo, were the best responding genotypes. Callus fragmentation and two subsequent transfers onto fresh medium at 7-day intervals yielded a frequency of plant regeneration of some 25–40 plantlets per gram fresh weight callus in 21 days on Murashige and Skoog's hormone-free medium. Plantlets could be efficiently established in soil, thus confirming the possibility of biotechnological approaches with varieties of this crop species.Abbreviations E embryogenic - NE non embryogenic - MS Murashige and Skoog's (1962) medium - 2,4-D 2,4 dichlorophenoxyacetic acid - DAA days after anthesis - FWT fresh weight tissue     

Somatic cell genetic studies inBrassica species

In vitro culture ofBrassica alba anthers on a growth medium containing inorganics of KB5 and organics, iron, sucrose and hormones of B5 resulted in a very high response of anthers (93.75%) towards callus induction. All the calli transferred to regeneration media responded favourably even after six months of callus induction. Numerous torpedo-shaped embryoids developed in clusters at many sites from each callus mass. Secondary embryogenesis and multiple shoot formation was also observed in many cases. The number of embryoids and plantlets produced by one embryogenic anther were as high as 169.8 and 17 respectively. 87% of the regenerated plants were haploids.     

Efficient somatic embryogenesis in sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) breeding lines

Efficient regeneration via somatic embryogenesis (SE) would be a valuable system for the micropropagation and genetic transformation of sugar beet. This study evaluated the effects of basic culture media (MS and PGo), plant growth regulators, sugars and the starting plant material on somatic embryogenesis in nine sugar beet breeding lines. Somatic embryos were induced from seedlings of several genotypes via an intervening callus phase on PGo medium containing N⁶-benzylaminopurine (BAP). Calli were mainly induced from cotyledons. Maltose was more effective for the induction of somatic embryogenesis than was sucrose. There were significant differences between genotypes. HB 526 and SDM 3, which produced embryogenic calli at frequencies of 25–50%, performed better than SDM 2, 8, 9 and 11. The embryogenic calli and embryos produced by this method were multiplied by repeated subculture. Histological analysis of embryogenic callus cultures indicated that somatic embryos were derived from single- or a small number of cells. 2,4-dichlorophenoxyacetic acid (2,4-D) was ineffective for the induction of somatic embryogenesis from seedlings but induced direct somatic embryogenesis from immature zygotic embryos (IEs). Somatic embryos were mainly initiated from hypocotyls derived from the cultured IEs in line HB 526. Rapid and efficient regeneration of plants via somatic embryogenesis may provide a system for studying the molecular mechanism of SE and a route for the genetic transformation of sugar beet.     

In vitro propagation of reed grass by somatic embryogenesis

A micropropagation system using regeneration via somatic embryogenesis from immature inflorescences has been optimized. This system is proposed for the production of the macrophyte Phragmites australis (Cav.) Trin. for the construction of wetlands used in wastewater purification. Embryogenic calli were produced in florets from inflorescences in the presence of 2,4-dichlorophenoxyacetic acid in the induction media. Up to 28.4% of the calli were embryogenic. Somatic embryos developed into plantlets when transferred to the regeneration medium lacking growth regulators. The addition of myo-inositol to the induction medium resulted in the highest number of plantlets on the regeneration medium. A decrease in the number of plantlets was observed when the embryogenic calli were maintained longer than three months on the induction medium. Plantlets can be further propagated by node culture. Plantlets were successfully acclimatized and developed normally showing no morphological differences when compared to seed-grown plants. This revised version was published online in June 2006 with corrections to the Cover Date.