Simple hormonal regulation of somatic embryogenesis and/or shoot organogenesis in caryopsis cultures of Pogonatherum paniceum (Poaceae)

Pogonatherum paniceum (Poaceae) is a perennial plant with good potential for eco-recovery and ornamental function. This study presents in vitro culture systems of simple hormonal regulation of somatic embryogenesis and shoot organogenesis from mature caryopses. Mature caryopses of P. paniceum were grown on Murashige and Skoog medium with 3% sucrose (w/v) and various concentrations or combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). Morphological development was analyzed by light microscope after histological sectioning. Four types of callus were induced by different concentrations of 2,4-D. Type I callus was regenerated via somatic embryogenesis; type II callus failed to produce any regeneration; type III callus had both somatic embryogenesis and shoot organogenesis capacities; and type IV callus only displayed shoot organogenesis capacity. Regarding hormone combinations used in this study, NAA only induced type IV callus and BAP only induced direct multiple shoot formation. The combinations of 2,4-D and NAA induced type III callus. Several of the regeneration pathways were simply controlled by one or two kinds of plant hormones. The established systems will be helpful for further research on the developmental mechanism of switch between somatic embryogenesis and shoot organogenesis.     

Histology of somatic embryogenesis in cultured leaf segments of sugarcane plantlets

Calli have been initiated from young leaves of in vitro grown sugarcane shoots. Histological examination has shown that the two types of calli induced (nodular and friable) originated from different regions of the explants and were cytologically different.This study has shown an obvious relation between the developmental stage of the excised tissue and the potential of plant regeneration of the in vitro initiated callus culture. Nodular calli were obtained from bases of the fast-growing young leaves while their more mature parts of the older leaves only produced friable calli. High-frequency plant regeneration via somatic embryogenesis was obtained from nodular calli while friable calli rarely produced plantlets.     

Genetic variability analyses of the somatic embryogenesis induction process in Olea spp. using nuclear microsatellites

The crop species Olea europaea L. (olive tree) is of great economic importance in the Mediterranean region. Hence, many efforts have been done in the last decades to propagate this commercially valuable species by in vitro methods. On the other hand, the lesser known Olea maderensis (Lowe) Rivas Mart. & Del Arco which is a native species of the Madeira Archipelago has only been the subject of micropropagation from nodal stem cuttings. Therefore, in this work we analysed the stability of ten nuclear simple sequence repeat (SSR) markers at successive stages of the somatic embryogenesis process in two adult trees belonging to these two species from the Madeira Archipelago. For the induction of somatic embryogenesis, petiole and leaf explants were cultivated on solid Murashige and Skoog medium (MS) with 12.25 μM indole-3-butyric acid (IBA) and 4.56 μM of zeatin, in the dark. After 3 months, different callus tissues (non-embryogenic, pre-embryogenic and embryogenic) developed from leaf explants and petioles were later transferred to MS medium without growth regulators in the dark. All ten SSR markers were able to distinguish between species. However, no mutations were found at the SSR loci at any of the successive developmental stages from PEMs (pre-embryogenic masses) to somatic embryos. This genetic uniformity was observed within material derived from each genotype/species and its respective donor plant. Therefore, we conclude that the genomic integrities of both O. europaea and O. maderensis were maintained throughout the stages of the embryogenic processes in study suggesting the absence of somaclonal variation.     

Auxin pretreatment promotes regeneration of sugarcane (Saccharum spp. hybrids) midrib segment explants

We have developed a new, simple, quick and genotype-independent method for direct regeneration of sugarcane using novel midrib segment explants. Our protocol involves two steps: the pretreatment of starting material on MS (Murashige and Skoog (1962) Physiol Plant 15:473–497) medium containing 3.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 8 days under continuous dark and subsequent transfer of the explants to MS medium augmented with 0.1 mg/l benzyladenine (BA) and 0.1 mg/l naphthaleneacetic acid (NAA) under light-dark conditions. On the regeneration medium, numerous globular structures appeared from the explants and subsequently differentiated into shoots. Regenerated shoots attained 2–5 cm height within 30 days of culture initiation and readily rooted on MS basal medium. Hardened plants were successfully established in the greenhouse. The regulation of sugarcane morphogenesis by auxin pretreatment is discussed.     

Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)

Somatic embryogenesis (SE) is a propagation tool of particular interest for accelerating the deployment of new high-performance planting stock in multivarietal forestry. However, genetic conformity in in vitro propagated plants should be assessed as early as possible, especially in long-living trees such as conifers. The main objective of this work was to study such conformity based on genetic stability at simple sequence repeat (SSR) loci during somatic embryogenesis in maritime pine (Pinus pinaster Ait.). Embryogenic cell lines (ECLs) subjected to tissue proliferation during 6, 14 or 22 months, as well as emblings regenerated from several ECLs, were analyzed. Genetic variation at seven SSR loci was detected in ECLs under proliferation conditions for all time points, and in 5 out of 52 emblings recovered from somatic embryos. Three of these five emblings showed an abnormal phenotype consisting mainly of plagiotropism and loss of apical dominance. Despite the variation found in somatic embryogenesis-derived plant material, no correlation was established between genetic stability at the analyzed loci and abnormal embling phenotype, present in 64% of the emblings. The use of microsatellites in this work was efficient for monitoring mutation events during the somatic embryogenesis in P. pinaster. These molecular markers should be useful in the implementation of new breeding and deployment strategies for improved trees using SE.     

Detection of microsatellite instability during somatic embryogenesis of oak (Quercus robur L.)

Five microsatellite loci (QpZAG1/5, QpZAG9, QpZAG36, MSQ4, MSQ13) were used to test for genetic stability of three somatic embryogenic culture lines of Quercus robur L. and plantlets derived therefrom. DNA variation was detected among somatic embryos within all embryogenic lines, whereas no genetic instability was found among the regenerated plants. Two microsatellite loci revealed variation, and a locus-dependent instability was observed. The most polymorphic and useful microsatellite locus for detecting genetic variation was QpZAG9, with 28.5% of the investigated loci being variable.     

Genotype and plant growth regulator-dependent response of somatic embryogenesis from Gentiana spp. leaf explants

Gentiana kurroo (Royle), Gentiana cruciata (L.), Gentiana tibetica (King. ex Hook. f.), Gentiana lutea (L.), and Gentiana pannonica (Scop.) leaves derived from axenic shoot culture were used as explants. For culture initiation, leaves from the first and second whorls from the apical dome were dissected and cultured on Murashige and Skoog (MS) basal medium supplemented with three different auxins: 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid (NAA), or 3,6-dichloro-o-anisic acid (dicamba) in concentrations of 0.5, 1.0, or 2.0 mg/l; and five different cytokinins: zeatin, 6-furfurylamonopurine (kinetin), N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ), N-(2-chloro-4-pyridyl)N′-phenylurea, or 6-benzylaminopurine (BAP). The cytokinin concentrations used were dependent on the type of cytokinin and varied between 0.25 and 3.0 mg/l. After 2 mo. of culture, the morphogenic response of explants was assessed. Frequency of embryogenesis was the highest for G. kurroo (54.7%) and dependent on plant growth hormones (PGRs). This gentian was the only species showing morphogenic capabilities on media supplemented with all applied combinations of PGRs, while none of the 189 induction media permutations stimulated somatic embryogenesis from G. lutea explants. G. tibetica and G. cruciata both produced an average of 6.6 somatic embryos per explant, while G. pannonica and G. kurroo regenerated at 15.7 and 14.2 somatic embryos per explant, respectively. Optimum regeneration was achieved in the presence of NAA combined with BAP or TDZ. This auxin also stimulated abundant rhizogenesis. Somatic embryos were also regenerated from adventitious roots of G. kurroo, G. cruciata, and G. pannonica. Somatic embryos converted into plantlets on half strength MS medium.     

Developmental patterns during direct somatic embryogenesis in protoplast cultures of european larch (Larix decidua Mill.)

Summary Protoplasts isolated from embryogenic suspension cultures of European larch (Larix decidua Mill.) were cultured in thin alginate layers using a nylon mesh to enable a monitoring of the development of single cells. The patterns of cell division and differentiation are characterized and compared with zygotic embryogenesis to which homologies can only be drawn to some extent when the protoplasts grow in an auxinfree environment. Already at 2.5 M both 2,4-dichlorophenoxyacetic acid or indole-3-acetic acid cause vacuolation and elongation of individual cells, thus disturbing the process of somatic embryogenesis which generally lacks the precise quantitative patterns occurring in vivo. Prior to the formation of an embryo, a proembryonal mass develops. Oligonucleated products of spontaneous protoplast fusions are able to cellularize even without preceding karyokinesis and perform a normal embryogenic program.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MES 2-(N-morpholino)ethanesulfonic acid - PEM proembryonal mass     

Cryopreservation of apices of in vitro plantlets of sugarcane (Saccharum sp. hybrids) using encapsulation/dehydration

Summary A cryopreservation process using encapsulation/dehydration was set up for apices sampled on in vitro plantlets of sugarcane. After dissection, apices were cultured for one day on standard medium and then encapsulated in medium with 3% alginate. Optimal conditions comprised preculture for 2 days in liquid medium with 250 g.l^–1 sucrose, desiccation for 6 hours under the laminar flow or for 10–11 hours with silicagel followed by rapid freezing and slow thawing. Survival after freezing in liquid nitrogen ranged between 38 and 91% for the 5 varieties experimented. Cryopreservation did not modify the electrophoretic profiles for aminoleucine peptidases and amylases with plants of the variety Co 6415.Abbreviations BAP 6-benzylaminopurine - KIN Kinetin - EDTA ethylenediamine tetracetic acid - AMP aminoleucine peptidases - AMY amylases - RFLP restriction fragment length polymorphism     

Plant regeneration through somatic embryogenesis in callus culture of green bamboo (Bambusa oldhamii Munro)

Summary Young inflorescence explants of green bamboo (Bambusa oldhamii Munro) in culture show a high capacity for plant regeneration through somatic embryogenesis. Embryogenic callus was initiated from explants maintained on Murashige and Skoog's medium supplemented with 3 mg/l 2,4-D, 2 mg/l kinetin and a high content (60 g/l) of sucrose. Prolonged culture in the embryoid induction medium or transferral of embryonic callus to auxin-free medium resulted in the continued development and eventual germination of embryoids and establishment of rooted plantlets that were successfully transferred to soil.     

Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.)

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 M thidiazuron but not on explants on media with benzyladenine (BA) or isopentenyladenine. Not all seed sources were equally capable of shoot organogenesis and embryogenesis. Atypical of adventitious regeneration of other woody plants, mature seed explants of white ash were more organogenic with shoots that elongated better than explants from immature seeds. Somatic embryogenesis was observed in cultures where mature seeds were first cultured for 4 weeks on a medium containing 10 M adenine 2,4-dichlorophenoxyacetic acid in combination with 0.1 and 1.0 M thidiazuron, followed by transfer to a medium containing 0.05 M 6-benzyladenine and 0.5 M naphthaleneacetic acid. Adventitious shoots and epicotyls from both seedlings and germinated somatic embryos were rooted under intermittent mist and acclimatized to the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - TDZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-ylurea - WPM woody plant medium     

Regeneration of pineapple plants via somatic embryogenesis and organogenesis

Summary We have developed efficient methods for plant regeneration, via both embryogenesis and organogenesis, of Smooth Cayenne pineapple, Ananas comosus (L.) Merr. Leaf bases and core (stem) sections of in vitro shoots, produced from culture of crown tip meristem, were used as explants for plant regeneration as follows: (1) Leaf base and core section explants cultured on Murashige and Skoog (MS) medium containing 41 μM 4-amino-3,5,6-trichloropicolinic acid (picloram, P) or thidiazuron (T)/P combinations produced embryogenic tissues. Different types of embryogenic tissues (friable emryogenic tissue, embryogenic cell cluster, and chunky embryogenic tissue) have been developed with varying properties in terms of growth rate and state of development. The embryogenic tissues regenerated shoots upon culture on MS medium containing 13 μM 6-benzylaminopurine (BA) and 1μM α-naphthaleneacetic acid (NAA) followed by culture on MS medium containing 4 μM BA. (2) Crown tip meristems cultured on MS medium containing 13 μM BA followed by leaf explants cultured on MS medium with 27 μM NAA and 1 μM BA produced shoots via direct organogenesis. (3) Explants cultured on MS medium containing 5 μM T and 0.5 μM indole-3-butyric acid (IBA) produced nodular globular structures, which produced shoots upon culture on MS medium containing 1 μM BA and 1 μM gibberellic acid. Shoots obtained from all of the above methods were rooted in half-strength MS medium containing 3 μM NAA and 2.5 μM IBA. Plants were transferred to the greenhouse or shipped to Costa Rica for field trials. Somatic embryo-derived plants exhibited 21 % spininess, and organogenic-derived plants exhibited 5% spininess in the field trials.     

The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses

Expression of the Agrobacterium rhizogenes rolC gene in Panax ginseng callus cells results in formation of tumors that are capable to form roots. The selection of non-root forming tumor clusters yielded the embryogenic 2c3 callus line, which formed somatic embryos and shoots independently of external growth factors. Although the 2c3 somatic embryos developed through a typical embryogenesis process, they terminated prematurely and repeatedly formed adventitious shoot meristems and embryo-like structures. A part of the shoots and somatic embryos formed enlarged and fasciated meristems. This is the first indication of the rolC gene embryogenic effect and, to our knowledge, the first indication that a single gene of non-plant origin can induce somatic embryogenesis in plants.     

In vitro regeneration of Echinacea purpurea L.: Direct somatic embryogenesis and indirect shoot organogenesis in petiole culture

Summary An in vitro propagation system was developed for Echinacea purpurea L. (purple coneflower), a medicinal plant commonly used in the treatment of colds, flu and related ailments. Echinacea seeds were found to be contaminated with systemic fungi and therefore an optimized minimal concentration of Plant Preservation Mixture (PPM) was incorporated in the seed germination medium to recover sterile seedlings. Regeneration was induced on petiole explants from 2-month-old sterile seedlings cultured on medium supplemented with benzylaminopurine (BAP) or thidiazuron (TDZ) in combination with indoleacetic acid (IAA). Two distinct forms of regeneration were identified in cultured petiole explants with histological and morphological observations, viz. the direct formation of somatic embryos on the epidermis and the de novo development of shoots from callus tissues formed in subepidermal cell layers. the results of this study have established a micropropagation system for E. purpurea that will provide sterile plant material for further investigations into medicinally active biochemicals and may facilitate mass production of high-quality E. purpurea plants for the commercial market.     

In vitro plant regeneration of Arachis correntina (Leguminosae) through somatic embryogenesis and organogenesis

In vitro protocols for plant regeneration of Arachis correntina through both somatic embryogenesis and organogenesis were developed using immature leaves as explants. Morphologically normal somatic embryos were obtained on culture media composed of 20.70 or 41.41 μM picloram (PIC) with the addition of 0.044 μM 6-benzylaminopurine (BA), resulting in a 33 and 24% of conversion into plants, respectively. The source of explants and the developmental stage of the leaves had a marked effect on somatic embryogenesis. The second folded immature leaves from in vitro growing plants were the most responsive producing up to 30% embryogenesis in MS+41.41 μM PIC. Embryos converted into plants after transfer to MS medium devoid of growth regulators and these plants were successfully acclimatised. Adventitious shoots were obtained on culture media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or naphthaleneacetic acid (NAA) with or without 0.044 μM BA, achieving plant regeneration in the induction media. The highest percentage of bud formation was obtained on culture medium composed of␣MS+10.74 μM NAA+0.044 μM BA (12.5%). Roots were formed on all culture media tested. Regenerated plants were transferred to pots and grew well under greenhouse conditions.     

Identification of somaclonal variants of sugarcane (Saccharum spp.) resistant to sugarcane mosaic virus via RAPD markers

Somaclonal variants resistant to sugarcane mosaic virus (SCMV) were obtained from susceptible sugarcane cv PR62258 through somatic embryogenesis by increasing the number of subcultures of the embryogenic callus tissue in MS medium with 3 mg/L 2,4-dichlorophenoxyacetic acid. Transfers were made at 30-day intervals for 1, 2 or 3 subcultures. Two somaclones, namely AT626 and BT627, were selected by their resistance to SCMV. These subclones have maintained the resistance trait over seven years of testing in the field. In this report we identified the somaclonal SCMV resistant variants from the maternal line and the nonresistant somaclones, using the RAPD technique.     

Phenotypic and karyotypic status of Beta vulgaris plants regenerated from direct organogenesis in petiole culture

Summary A method for high frequency in vitro regeneration from petiole explants was tested on nine breeding lines of Beta vulgaris L. from the haploid, diploid and tetraploid levels. Regenerants could be obtained without a callus step, from excised petioles derived either from axillary buds sprouted in vitro or from field grown plants, by plating the explants on MS medium supplemented with TIBA (2,3,5-triiodobenzoïc acid) and BAP (6-Benzylaminopurine). The multiple shoots obtained were then rooted in vitro and transferred to soil. In some cases, these adventitious shoots were also used as a petiole explant source for further petiole culture cycles, and the phenotypic characteristics and ploidy status of the regenerants were investigated after one or three petiole culture cycles. Conventional shoot apex culture was used as an in vitro control. Phenotypic variations such as differences in morphology and changes in in vitro growth behaviour, were noticed. Chloroplast and chromosome counts indicated that the alterations in morphogenetic pathway could not be explained by the occurrence of gross cytogenetic abnormalities such as aneuploidy or myxoploidy. Our results suggest that the altered morphology is caused by the presence of the exogenous antiauxin (TIBA) during the in vitro phase. Following transfer to the greenhouse, none of these variations persisted and cytogenetic analyses revealed karyotypic stability in all the plants studied, even after three petiole culture cycles. An assessment of the in vitro petiole culture method as a true-to-type multiplication method for Beta vulgaris is made.     

Callus culture of Coronilla varia L. (crownvetch): plant regeneration through somatic embryogenesis

Callus culture and plant regeneration through somatic embryogenesis have been obtained in Coronilla varia. Media used were UM (25) supplemented with 2 mg/l 2,4-D followed by subculture on MS (18) containing 1 mg/l 2-iP and 0.1 mg/l IAA. Embryoids developed into complete plantlets on filter paper saturated with hormone-free MS medium.