Somatic Embryo Formation Research Articles

High Frequency Plant Regeneration System from Transverse Thin Cell Layer Section of <i>In vitro</i> Derived ‘Nadia’ Ginger Microrhizome

An efficient and reproducible procedure is outlined for rapid in vitro multiplication of Zingiber officinale var. ‘Nadia’ through high frequency shoot proliferation from transverse thin cell layer (tTCL) sections of in vitro derived microrhizome. In vitro derived microrhizome of size 500 μm in thickness was used as initial explants for induction of somatic embryos. Among the different phytohormones tested, tTCL explants shows maximum calli proliferation in medium containing 2 mg/L 2,4-Dichlorophenoxyacetic acid (88.30±0.11%). Reduced concentration of 2,4 Dichlorophenoxyacetic acid was supplemented with different cytokinins for regeneration of callus. Among the different medium tested, optimum redifferentiation of somatic embryos were observed in medium containing 0.2 mg/L 2,4 Dichlorophenoxyacetic acid and 6.0 mg/L BAP (141.08±0.25). Clump of regenerated plantlets were further subculture and transfer into microrhizome inducing medium containing high sucrose concentration (8%). Plantlets with well developed microrhizome were successfully acclimatized and eventually transferred to the field. The application of studying embryo section for regeneration of plants might be useful alternative to ginger improvement programme. Histological analysis showed formation of somatic embryos and regenerated adventitious shoot.

ANTIOXIDANT ENZYMES ACTIVITY IN EMBRYOGENIC AND NON-EMBRYOGENIC TISSUES IN SUGARCANE

The objective of this work was to induce direct somatic embryogenesis from segments of immature leaves of the RB872552 variety of sugarcane and to correlate this morphogenic event with oxidative stress. Two previously described protocols were utilized for the induction of somatic embryogenesis in sugarcane with different supplementations of the culture medium and different incubation conditions. For the conversion of embryos into plants was used MS medium without phytoregulators. Histological analyses and activity of antioxidant enzymes were also conducted for the embryogenic and non-embryogenic tissues. The formation of somatic embryos was obtained in 81 % of the explants with the combination of regulators 2,4-D (2,4-dichlorophenoxyacetic acid)and BAP (6-benzylaminopurine) when incubated under 16 h photoperiod. With regards to the antioxidant enzymes, there was increased activity of peroxidase and an increase in the soluble protein content in embryogenic tissues, whereas lower activities of polyphenol oxidase and catalase appeared in these tissues compared to nonembryogenic tissues. It could be inferred that oxidative stress plays an important role in the induction of somatic embryogenesis in sugarcane.

HISTOLOGICAL ANALYSIS OF SOMATIC EMBRYOGENESIS INDUCED IN TROPICAL LADY'S SLIPPER ORCHID (PAPHIOPEDILUM NIVEUM)

Indirect somatic embryogenesis, an asexual process of plant propagation, is an acceptable tool for cell differentiation and developmental studies. This study was conducted to elucidate the developmental pathway of a somatic embryo in the Paphiopedilum niveum orchid. The callus-derived somatic embryo was obtained by using modified Vacin and Went (VW) medium supplemented with 0.1 mg L-1 naphthalene acetic acid (NAA) and 0.5 mg L-1 thidiazuron (TDZ) as well as 10 g L-1 sucrose. Developmental phases were characterized by histological analysis. The callus-derived somatic embryos developed progressively through the early globular, globular and cotyledonary stages. After a month of culture, a proembryonic mass (or early globular-shape stage) originated on the surface of the callus and differentiated rapidly to a globular somatic embryo with a well-demarcated protoderm. These somatic embryos developed protrusions that indicated the cotyledonary stages after two months of culture. The organized structures of plantlets were eventually obtained when protocorm-like bodies (PLBs) involving somatic embryogenesis were transferred to modified Murashige and Skoog (MS) medium supplemented with 50 g L-1 homogenated banana. Histological analysis also revealed that the pattern of somatic embryo formation of P. niveum showed the same process as the developmental pathway of monocot species.

Improvement Germination and Conversion of Somatic Embryos and Production of Normal Plantlets in Ferula Assa-foetida L. (A Rare Medicinal Plant)

Secondary somatic embryogenesis leads to the formation of abnormal somatic embryos and produces abnormal seedlings. Normal plants are difficult to obtain from these embryos, due to the asynchronous maturationof the embryogenic tissues and low germination and conversion rates. The effects of some media additives and different strengths of MS medium on germination and plantlet formation of in vitro derived somatic embryos of FerulaAssa-foetidawere studied.The highest number of normal embryos was observed in MS medium containing 30g/l sucrose with 0.5% or 1% AC and in MS medium supplemented with PEG and 0.5% or 1% AC. The treatments of MS medium with 30g/l sucrose and 0.5% AC ◊ MS medium containing sorbitol and MS medium containing PEG and 1% AC ◊ Ω MS had maximum number of normal germinated embryos without secondary somatic embryogenesis (SSE). In some of the treatments the embryos were converted better than the others, such as; the interact ion effect of MS medium with 30g/l sucrose and0.5% AC◊ MS, MS medium with 30g/l sucrose ◊ MS medium with glutamine. Using different strength of MS medium and presence of some media additives is effective on germination and conversion of somatic embryos into normal plantlet. Presence of Activated Charcoal in the culture medium can reduce secondary somatic embryogenesis.

English

Jatropha curcas L. is an important non-edible oil yielding plant growing in wasteland and dry lands. The experiment was conducted to study the plant regeneration of J. curcas from different ages of petioles. Petioles explant grown on Murashige and Skoog’s (MS) medium supplemented with 1 mg/l α-naphthalene acetic acid and 0.2 mg/l kinetin showed the highest frequency of callus induction and the same medium was found to be best suited for callus growth in 1st petiole. Percent of somatic embryo formation was higher (35.16%) from the 1st petiole explant on 0.5 mg/l thidiazuron with 0.4 mg/l gibberellic acid. MS medium supplemented with 1 mg/l N6-benzylaminopurine, 0.5 mg/l indole-3-acetic acid, 0.25 mg/l kinetin recorded the higher somatic embryo germination percentage (66.85%). MS basal medium supplemented with 0.5 mg/l thidiazuron, 0.1 mg/l N6-benzylaminopurine, 0.4 mg/l gibberellic acid was found to be best medium for shoot elongation. Among the auxins tested, the higher frequency of root formation was observed in MS medium supplemented with 0.3 mg/l α-naphthalene acetic acid compared to indole-3-butyric acid and indole-3-acetic acid. The survival rate of in vitro rooted plantlets was dependent on the type of explant source and it varies from 47 to 60%.   Key words: Euphorbiaceae, petiole, callus, indirect organogenesis, biodiesel, plant growth regulator.&nbsp

Influence of growth regulators on callus induction from embryos of five citrus rootstocks

Objective: The aim of this work is to develop a protocol for callus induction of Moroccan Citrus rootstocks. Methodology and results: Callus cultures were initiated from embryos explants of five Citrus rootstocks (Cleopatra mandarin , Rangpur lime , Citrus volkameriana , Trifoliate orange , Citrus aurantium ) on Murashige and Tucker (MT) and Tucker (MT) basal media. Different concentrations of growth regulators were tested in order to obtain the best callus formation: 2, 4-dichloro-phenoxyacetic acid (2, 4-D) in combination with benzylaminopurine (BAP) at five levels (0, 0.5, 1, 2 and 3mgL-1) were used in this study. It was found that growth regulator concentration had a significant effect on the callus induction, the callus growth and callus physical appearance. The highest frequency of the callus induction rate (100% and 83%) was observed with two combinations of 2, 4-D/BAP: 1 / 0, 5 and 2/1 (mgl-1). Medium containing only BAP (1mgl-1) resulted in the formation of large numbers of roots. Also, the callus induced on MT medium containing only 2, 4-D (1mgl-1) was brown in color and of low quality compared to that produced on MT media containing 2, 4-D/BAP. There was no callus formation on MT basal medium without growth regulators. Also, the callogenesis depended on the genotype. It was maximal for Cleopatra mandarin followed by Rangpur lime and Citrus volkameriana , and then Citrus aurantium and trifoliate orange . Conclusion and application : Passing through callus is paramount in vitro selection that plant tissue is an important source of genetic variability. The explants and exogenous phytohormones proved of paramount importance for the success of the callus and the formation of somatic embryos and their development.

Development of Insect Resistant Transgenic Lines Mediated by <i>Agrobacterium</i> in Cotton (<i>Gossypium hirsutum</i> L.)

In order to produce transgenic cotton resistant to insects, five months old embryogenic calli of Coker 310 were transformed with Agrobacterium tumefaciens strain LBA4404 harbouring the plasmid pBI 121 containing cry2Ab gene under the control of CaMv 35S promoter. Neomycin phosphotransferase (nptII) gene was used as a selectable marker. Infected embryogenic calli were incubated in dark at 24°C in co-cultivation medium containing 100mM acetosyringone for three days. Transformed calli were selected on selection medium containing 25 mg/L kanamycin and 500 mg/L cefotaxime. After four rounds of selection (each round with 15 days duration) the induction of kanamycin resistant embryogenic calli was at an average frequency of 37.0% and further two subcultures (each with one month duration) of the kanamycin resistant embryogenic calli in the same media resulted in the formation of somatic embryos at an average frequency of 23.7%. The matured cotyledonary stage embryos were germinated on the plantlet development medium without kanamycin selection. Out of 500 explants co-cultivated, 5 plants (1.0% transformation efficiency) showed the presence of genes nptII and cry2Ab through PCR analysis. Insect bioassay against Helicoverpa armigera performed with the five T 0 plants showed 33.0% to 67.0% larval mortality.

Proteomic analysis and polyamines, ethylene and reactive oxygen species levels of Araucaria angustifolia (Brazilian pine) embryogenic cultures with different embryogenic potential

Somatic embryogenesis is an important biotechnological tool in the large-scale propagation of elite genotypes and ex situ conservation of conifer species. Protocols for the induction and proliferation of embryogenic cultures (ECs) of Brazilian pine (Araucaria angustifolia (Bert.) O. Ktze) are well established, although the proper formation of mature somatic embryos (SEs) is still problematic. Thus, the identification of molecular markers for the screening of ECs able to respond to maturation conditions (abscisic acid and osmotic agents) is highly desirable. To develop molecular markers for the early detection of ECs able to develop well-formed SEs under maturation conditions, we analyzed the proteins found during the proliferation phase of A. angustifolia cell lines with different embryogenic capabilities, with one cell line being responsive to maturation conditions (R cell line), and one cell line that presented blocked development of SEs (B cell line). In addition, based on the peptides identified, polyamine levels (free and conjugate), ethylene production and reactive oxygen species (ROS) emission were analyzed using both EC lines (R and B cell lines). A marked difference in the biochemistry of ECs between these two cell lines was observed. Eleven proteins that were differentially expressed in the cell lines were identified by the combination of two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry. Among these, S-adenosylmethionine synthase, the enzyme associated with polyamines and ethylene biosynthesis, was observed exclusively in the R cell line, while a protein linked to the oxidative stress subunit F of NADH dehydrogenase was observed exclusively in the B cell lines. Additionally, B cell lines showed higher levels of diamine putrescine and lower levels of ethylene. Higher values of ethylene and ROS were observed for the cell line that showed normal development of SEs. Altogether, our results open new perspectives in the optimization of culture conditions for A. angustifolia somatic embryogenesis, as well as establishing biochemical markers for the early selection of ECs during maturation trials.

The effects of growth regulators and a scanning electron microscope study of somatic embryogenesis in Antartic hair grass (Deschampsia antarctica Desv.)

Deschampsia antartctica Desv. is a type of grass that is physiologically and biochemically adapted to the extreme environmental conditions of the Antarctic continent, which is of interest to many investigators. To explore the potential use of somatic embryogenesis as a biotechnological tool for the mass micropropagation of this grass, the effects of three dosages of 2,4-dichlorophenoxyacetic acid, dicamba, and picloram were evaluated. The developmental and morphological stages of somatic embryo formation were evaluated using scanning electron microscopy (SEM). Plant regeneration was evaluated under the effects of different dosages of 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA), alone and combined. The results indicated that a Murashige and Skoog basal medium supplemented with 3 mg/l of dicamba was the best for inducing somatic embryogenesis, while the combination of 1 mg/l BAP and 0.1 mg/l of NAA was the most efficient for the regeneration and development of the plants. This work demonstrates, for the first time with the use of SEM, that it is possible to apply somatic embryogenesis for the regeneration of superficial and morphological structures of somatic embryos in the species D. antarctica.

INDUKSI EMBRIO SOMATIK Shorea pinanga Scheff. PADA KONDISI FISIK MEDIA BERBEDA

Shorea pinanga (Scheff.) is a member of Dipterocarpaceae which has an important role both as a forest timber and also non forest timber product (tengkawang). The purpose of this experiment is to obtain the best treatment for somatic embryos formation. Friable callus was used as explants whereas liquid and solid MS media supplemented with vitamin B, 30 g/l sucrose were used as basal medium. Embryogenic callus was induced through three times subcultured in basal medium with 5.0 mg/l 2,4- D. Somatic embryo was induced using 100 mg of embryogenic callus with the addition of 0.5, 1.0, and 1.5 mg/l kinetin. The observation was done on texture, numbers and color of embryogenic callus and weekly number of somatic embryos for eight weeks. The kinetin treatment of 1.5 mg/l on liquid medium was the best treatment because it is able to induce 162 cotyledonary stage of somatic embryos for eight weeks.

Induced Expression of AtLEC1 and AtLEC2 Differentially Promotes Somatic Embryogenesis in Transgenic Tobacco Plants

Arabidopsis LEAFY COTYLEDON (LEC) genes, AtLEC1 and AtLEC2, are important embryonic regulators that play key roles in morphogenesis and maturation phases during embryo development. Ectopic expression of AtLEC1 and AtLEC2 in tobacco caused abnormality in transgenic seedling. When transgenic seeds germinated on medium containing 30 µM DEX, LEC1 transgenic seedlings were ivory and fleshy, with unexpanded cotyledons, stubby hypocotyls, short roots and no obvious callus formation at the shoot meristem position. While LEC2 transgenic seedlings formed embryonic callus on the shoot apical meristem and somatic embryo-like structures emerged from the surface of the callus. When callus were transferred to hormone free MS0 medium more shoots were regenerated from each callus. However, shoot formation was not observed in LEC1 overexpressors. To investigate the mechanisms of LEC2 in somatic embryogenesis, we studied global gene expression by digital gene expression profiling analysis. The results indicated that ectopic expression of LEC2 genes induced accumulation of embryo-specific proteins such as seed storage proteins, late embryogenesis abundant (LEA) proteins, fatty acid biosynthetic enzymes, products of steroid biosynthesis related genes and key regulatory genes of the embryo development. Genes of plant-specific transcription factors such as NAC domain protein, AP2 and GRAS family, resistance-related as well as salicylic acid signaling related genes were up-regulated in LEC2 transgenic seedlings. Ectopi c expression of LEC2 induced large number of somatic embryo formation and shoot regeneration but 20 d DEX induction of LEC1 is not sufficient to induce somatic embryogenesis and shoot formation. Our data provide new information to understand the mechanisms on LEC2 gene’s induction of somatic embryogenesis.

Biochemical status of in vitro regenerated Lilium bosniacum and Lilium cattaniae plantlets

Abstract Lilium cattaniae (Vis.) Vis. and Lilium bosniacum (G. Beck) Beck ex Fritsch, endemic species of Balkan Dinaric Alps, were micropropagated from seeds collected from their natural habitats. The relationship between peroxidase activity, photosynthetic status and differentiation of Lilium cattaniae and L. bosniacum in vitro was investigated. Peroxidase activity recorded for somatic embryos of Lilium cattaniae obtained on Murashige and Skoog (MS) medium containing 9.05 mM 2.4-dichlorophenoxyacetic acid (2.4-D) and 4.44 mM N6-bezyladenin (BA), was about two times higher than for any other treatment. Photosynthetic status of plantlets obtained through regeneration was explant-specific and generally higher for plantlets regenerated from basal leaf explants than from bulb explants. The accumulation of anthocyanin was detected in some regenerated shoots and more often in plantlets obtained through regeneration from bulb explants. High frequency of somatic embryo formation was recorded for L. cattaniae on MS medium containing 9.05 mM 2.4-D and 4.44 mM BA. The peroxidase activity for L. bosniacum plantlets obtained through regeneration showed treatment-specific elevations. We consider that basal leaf parts are suitable for successful regeneration of these two lilies and that elevation in peroxidase activity is a good indicator of somatic embryogenesis in both lilies.

Superoxide dismutase multigene family in longan somatic embryos: a comparison of CuZn-SOD, Fe-SOD, and Mn-SOD gene structure, splicing, phylogeny, and expression

Superoxide dismutases (SODs), which are encoded by a multigene family, are important antioxidant enzymes guarding against superoxide toxicity. Because no systematic characterization of this gene family has been conducted in plant embryos, the function of SODs in longan embryos is not completely clear. In this study, we isolated 20 full-length cDNAs encoding cytoplasmic CSD1a and DlCSD1b, chloroplastic DlCSD2a and DlFSD1a, plastidic DlFSD1b, and mitochondrial DlMSD from longan embryogenic callus using reverse transcription-PCR and rapid amplification of cDNA ends. Each gene possessed multiple polyadenylation sites. The DlCSD1a, DlCSD1b, DlCSD2a, DlFSD1a, DlFSD1b, and DlMSD genes contained eight, six, seven, six, seven, and five introns, respectively, of greatly varying length. Seven variants with different splicing modes, demonstrating functional diversity during longan somatic embryogenesis (SE), were also cloned. Each SOD gene type had multiple transcription start sites (TSSs). The choice of TSS affected the length of the 5′-untranslated region but did not contribute to protein diversity. DlCSD1a, DlCSD2a, DlFSD1a, and DlMSD promoters were isolated and found to contain many cis-acting elements responsive to stimuli such as light, gibberellins, auxin, methyl jasmonate, dehydration, coldness, and wounding. Comprehensive expression profile analysis indicated that different SOD types exhibit different spatial and temporal expression modes and play a key role in longan SE, especially during the middle and later developmental stages. This study represents the first comprehensive analysis of the SOD gene family in plant SE, encompassing full-length cDNA cloning, gene structures, alternatively spliced variants, phylogeny, TSS, promoter isolation, and expression patterns. The comparisons in this study provide insights into diverse functions of SOD isoforms during longan somatic embryo formation.

LEAFY COTYLEDON2 (LEC2) promotes embryogenic induction in somatic tissues of Arabidopsis, via YUCCA-mediated auxin biosynthesis

The LEAFY COTYLEDON2 (LEC2) transcription factor with a plant-specific B3 domain plays a central role in zygotic and somatic embryogenesis (SE). LEC2 overexpression induced in planta leads to spontaneous somatic embryo formation, but impairs the embryogenic response of explants cultured in vitro under auxin treatment. The auxin-related functions of LEC2 appear during SE induction, and the aim of the present study was to gain further insights into this phenomenon. To this end, the effect of LEC2 overexpression on the morphogenic responses of Arabidopsis explants cultured in vitro under different auxin treatments was evaluated. The expression profiles of the auxin biosynthesis genes were analysed in embryogenic cultures with respect to LEC2 activity. The results showed that LEC2 overexpression severely modifies the requirement of cultured explants for an exogenous auxin concentration at a level that is effective in SE induction and suggested an increase in the auxin content in 35S::LEC2-GR transgenic explants. The assumption of an LEC2 promoted increase in endogenous auxin in cultured explants was further supported by the expression profiling of the genes involved in auxin biosynthesis. The analysis indicated that YUCCAs and TAA1, working in the IPA-YUC auxin biosynthesis pathway, are associated with SE induction, and that the expression of three YUCCA genes (YUC1, YUC4 and YUC10) is associated with LEC2 activity. The results also suggest that the IAOx-mediated auxin biosynthesis pathway involving ATR1/MYB34 and CYP79B2 does not seem to be involved in SE induction. We conclude that de novo auxin production via the tryptophan-dependent IPA-YUC auxin biosynthesis pathway is implicated in SE induction, and that LEC2 plays a key role in this mechanism.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-013-1892-2) contains supplementary material, which is available to authorized users.

Relationships between endogenous hormonal content and direct somatic embryogenesis in Prunus persica L. Batsch cotyledons

Cotyledons of peach (Prunus persica L. Batsch cv. ZiseMay®) were cultured in vitro on medium deprived of plant growth regulators. Two different lines varying in their embryogenic capacity were studied after 90 days in culture media. Endogenous levels of abscisic acid (ABA), indole-3-acetic acid (IAA), trans-zeatin (Z), trans-zeatin riboside (ZR), the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), salicylic acid (SA), and jasmonic acid (JA) were analyzed in embryogenic and non-embryogenic cotyledons. No significant differences were observed in total ABA, IAA, ZR, SA and JA concentrations between the embryogenic and non-embryogenic cotyledons. On the contrary, lower Z and ACC contents, and also a reduced balance between Z and IAA levels were related with the embryogenic capacity of the cotyledons. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic and non-embryogenic cotyledons is related to their endogenous Z contents, and that the endogenous hormonal balance between Z and IAA is an important index defining the embryogenic potential in peach cotyledons.

SOMATIC EMBRYOGENESIS IN SEED LEGUMES: THE POSSIBLE ROLE OF SOLUBLE AUXIN RECEPTORS

ABSTRACT The formation of somatic embryos in higher plants in vitro is an exogenously regulated process which can be divided into (a) the induction phase and (b) the release of induced cells for further development. There are, however, many examples in the literature where induction of somatic embryogenesis is restricted to certain genotypes or cultivars. The solution for overcoming the regeneration problem in these cases requires laborious and time-consuming try-and-error approaches, including the testing of numerous media and growth conditions, with unpredictable success. Since in most plant species, including cereals and seed legumes, the induction of somatic embryogenesis is a strictly auxin-controlled process, we studied the signal transduction pathway, focusing on the primary interaction site, namely auxin recognition. This requires at the beginning the characterization of specific auxin receptors in explants exhibiting embryogenic potential. In immature zygotic pea embryos and shoot tips of etiolat...

Characterization of genes associated with polyembryony and in vitro somatic embryogenesis in Citrus

Apomixis is a common reproduction system in the genus Citrus. To investigate the molecular mechanism of apomictic embryogenesis in Citrus, genes expressed specifically in an apomictic genotype were isolated by PCR-selected suppression subtractive hybridization with total RNAs obtained from the ovule at anthesis. Several genes showing conspicuously different expressions between polyembryonic (apomictic) and monoembryonic (nonapomictic) genotypes were selected, and their expression profiles during ovule development were analyzed in detail. This analysis identified two apomictic and three nonapomictic genotype-specific genes. Among the latter, msg-2 was highly expressed in the late stage of somatic embryogenesis. Specific expression during ovule development in monoembryonic cultivars and in the late stage of somatic embryogenesis indicated that msg-2 is not expressed in the initiation stage of polyembryogenesis and somatic embryogenesis, suggesting its role in suppressing initial cell formation of somatic embryos. The full-length complementary DNA of msg-2 contained small open reading frames in its sequence but showed no homology to functionally known genes in the public databases. As sequences similar to msg-2 were frequently found among Citrus expressed sequence tags, msg-2 may be associated with polyembryogenesis and somatic embryogenesis in a Citrus-specific manner.

A comparative study between solid and liquid cultures relative to callus growth<br>and somatic embryo formation in date palm (Phoenix dactylifera L.) cv. Zaghlool

Tissue culture techniques enable mass propagation of elite cultivars of date palm (Phoenix dactylifera L.). The main limitations for date palm in vitro multiplication are the low rates achieved using solidified medium and the long period needed to produce acclimatized plantlets. This research focuses on the comparison between different culture types and plant growth regulator (PGR) combinations on callus growth and somatic embryo formation of cv. Zaghlool. For callus growth, 200 mg friable embryogenic callus dispensed in Rasotherm and Phytacon flasks containing 200 ml liquid (100 rotations per minute) and in temporary immersion system, RITA ® bioreactor (5 min immersion every 12 h), were compared with cultures grown on 200 ml solidified medium. For somatic embryo formation, 500 mg of friable embryogenic callus grown in Erlenmeyer flasks filled with 50 ml liquid or solid MS medium. The medium was supplemented with 0.1 mg l -1 Naphthaleneacetic acid (NAA), 1.5 g l -1 activated charcoal (AC) with or without 0.05 mg l -1 6-Benzyl amino purine (BAP) compared to PGR-free medium. Results proved that cell suspension cultures produced the highest callus fresh mass as compared to the other systems tested, and the callus fresh mass reached 4 g after 16 weeks. The temporary immersion system did not significantly enhance the fresh mass of callus compared to the solidified medium. For somatic embryo induction, the number of somatic embryos increased in cell suspensions 6-16 fold compared to the solidified medium. Using liquid MS medium enriched with 0.1 mg l -1 NAA and 1.5 g l -1 AC gave rise to the highest number of somatic embryos formed from 500 mg initial callus: 160 embryos. The number of somatic embryos was also affected by the callus source. The calli induced from leaflet segments excised from converted somatic embryos resulted in a lower somatic embryo number than those of shoot tip origin with about 60 somatic embryos per 500 mg callus. The formation of somatic embryos using liquid medium required only 6 weeks, thus considerably reducing the previously reported period of 18 weeks which is required for somatic embryo formation using solidified media.

Molecular overview on plant somatic embryogenesis.

Abstract The developmental pathway leading to plant somatic embryogenesis (SE) is true demonstration of totipotency of plant cells. During this process, somatic cells, under appropriate conditions, divide and differentiate into embryos. This developmental pathway plays an important role as an efficient means for plant regeneration and large-scale propagation. It includes a profound reprogramming of gene expression leading to changes in cell division and differentiation patterns, becoming a suitable platform to study the morpho-physiological and molecular aspects involved in plant cell differentiation and embryo development. Plant growth regulators such as auxin, as well as stress factors and DNA methylation, are key components to induce entry into SE pathways. Proteome and transcriptome analysis allowed isolation and characterization of embryogenic-specific gene markers involved in promoting vegetative-to-embryogenic transition as well as in maturation of somatic embryos contributing to the understanding of complex relationships between inductive conditions and somatic embryo formation. This review describes current advances made, mainly at the molecular level, in discovery of the main factors involved in the induction and maturation of somatic embryos providing a basic background for understanding genetic reprogramming that is at the heart of this process. We paid special attention to extracellular protein markers during SE as well as to auxin, abscisic acid and ethylene response genes, transcriptor factors and proteins involved in embryogenic competence acquisition.

Стволовые клетки побега растений – генетическая регуляция

This article describes the main features of plant stem cells and summarizes the results of studies of the genetic control of stem cell maintenance in the apical meristem of the shoot. It is demonstrated that the WUS-CLV gene system plays a key role in the maintenance of shoot apical stem cells and the formation of adventitious buds and somatic embryos. Unconventional concepts of plant stem cells are considered.