Induction Of Somatic Embryogenesis Research Articles

Recovery of virus-free Almond (Prunus dulcis) cultivars by somatic embryogenesis from meristem undergone thermotherapy

One of the world's main horticulture problems is the contamination of fruit trees with a variety of plant diseases, especially viral and pseudo-viral diseases. Due to the non-sexual propagation of the trees, these diseases have been transmitted to different parts of the world. The main aim of this study was to obtain a new effective method for virus elimination from almond cultivars, which was performed in two phases. In the first phase, we tested various almond cultivars with ELISA and RT-PCR. The results showed the infection of mother plantlets. So, three types of in vitro thermotherapy treatments were performed on infected plants to make them virus-free. The plantlets obtained from 0.5 mm meristem treated with the first type of thermotherapy (TH1: 8 h at 27 °C and 16 h at 38 °C for 18 days) showed the highest percentage of elimination of ApM, ACLS and TRS viruses. In the second phase, meristems were cultured on MS medium containing 0, 0.5, 1 and 2 mg/L 2,4-D with 1 mg/L TDZ and after two weeks, thermotherapy treatments were performed. The results showed, combining three methods of thermotherapy (TH1), meristem culture and somatic embryogenesis induction from meristem on MS medium supplemented with 0.5 mg/L 2,4-D and 1 mg/L TDZ is the most effective and safe technique for virus eradication without meristem size challenges. The samples that were diagnosed as virus-free were proliferated in temporary immersion bioreactor systems, and rooted to be used for later propagation and establishment of mother healthy orchards.

Effect of Different Cytokinins on Shoot Outgrowth and Bioactive Compounds Profile of Lemograss Essential Oil

Lemongrass (Cymbopogon citratus) essential oil (EO) is a major source of bioactive compounds (BC) with anticancer activity such as α-citral, limonene, geraniol, geranyl acetate, and β-caryophyllene. Comparative studies about cytokinin effects on BC profiles in lemongrass are missing. Here, we evaluated four cytokinins (2iP, tZ, BAP, and KIN) in two different osmotic media, MS-N (3% sucrose, 3 g L−1 Gelrite™) and MS-S (5% sucrose, 5 g L−1 Gelrite™). It results in a higher multiplication rate in BAP containing medium compared to tZ, KIN, and 2iP (p ≤ 0.05). While shoots grown on MS-N/BAP, tZ, and KIN exhibited a highly branching morphology, MS-N/2iP produced a less branching architecture. BC profile analysis of established plants in pots revealed that their maxima production depends on the in vitro shoot growth conditions: i.e., highest content (80%) of α-citral in plants that were cultured in MS-S/BAP (p ≤ 0.05), limonene (41%) in MS-N/2iP, or geranyl acetate (25.79%) in MS-S/2iP. These results indicate that it is possible to increase or address the production of BC in lemongrass by manipulating the cytokinin type and osmotic pressure in culture media. The culture protocol described here is currently successfully applied for somatic embryogenesis induction and genetic transformation in lemongrass.

Dynamic Transcriptome Analysis Reveals Complex Regulatory Pathway Underlying Induction and Dose Effect by Different Exogenous Auxin IAA and 2,4-D During in vitro Embryogenic Redifferentiation in Cotton

Plant somatic cells can reprogram into differentiated embryos through somatic embryogenesis (SE) on the condition of plant growth regulators (PGRs). RNA sequencing analysis was performed to investigate transcriptional profiling on cotton redifferentiated callus that was induced by different auxin types (IAA and 2,4-D), different concentrations (0, 0.025, and 0.05 mg L−1), and different incubation times (0, 5, and 20 days). Under the 2,4-D induction effect, signal transduction pathways of plant hormones were significantly enriched in the embryogenic response stage (5 days). These results indicated that auxin signal transduction genes were necessary for the initial response of embryogenic differentiation. In the pre-embryonic initial period (20 days), the photosynthetic pathway was significantly enriched. Most differentially expressed genes (DEGs) were downregulated under the induction of 2,4-D. Upon the dose effect of IAA and 2,4-D, respectively, pathways were significantly enriched in phenylpropanoid biosynthesis, fatty acid metabolism, and carbon metabolic pathways. Therefore, primary and secondary metabolism pathways were critical in cotton SE. These results showed that complex synergistic mechanisms involving multiple cellular pathways were the causes of the induction and dose effect of auxin-induced SE. This study reveals a systematic molecular response to auxin signals and reveals the way that regulates embryogenic redifferentiation during cotton SE.

Induction of Somatic Embryogenesis in Tamarillo (Solanum betaceum Cav.) Involves Increases in the Endogenous Auxin Indole-3-Acetic Acid.

Somatic embryogenesis (SE) is a complex biological process regulated by several factors, such as the action of plant growth regulators, namely auxins, of which the most physiologically relevant is indole-3-acetic acid (IAA). In tamarillo, an optimized system for induction of SE creates, after an induction process, embryogenic (EC) and non-embryogenic callus (NEC). In this work the endogenous levels of auxin along the induction phase and in the calli samples were investigated using chemical quantifications by colorimetric reactions and HPLC as well as immunohistochemistry approaches. Differential gene expression (IAA 11, IAA 14, IAA 17, TIR 1, and AFB3) analysis during the induction phase was also carried out. The results showed that the endogenous IAA content is considerably higher in embryogenic than in non-embryogenic calli, with a tendency to increase as the dedifferentiation of the original explant (leaf segments) evolves. Furthermore, the degradation rates of IAA seem to be related to these levels, as non-embryogenic tissue presents a higher degradation rate. The immunohistochemical results support the quantifications made, with higher observable labeling on embryogenic tissue that tends to increase along the induction phase. Differential gene expression also suggests a distinct molecular response between EC and NEC.

One-Week Scutellar Somatic Embryogenesis in the Monocot Brachypodium distachyon.

Plant somatic embryogenesis (SE) is a natural process of vegetative propagation. It can be induced in tissue cultures to investigate developmental transitions, to create transgenic or edited lines, or to multiply valuable crops. We studied the induction of SE in the scutellum of monocots with Brachypodium distachyon as a model system. Towards the in-depth analysis of SE initiation, we determined the earliest stages at which somatic scutellar cells acquired an embryogenic fate, then switched to a morphogenetic mode in a regeneration sequence involving treatments with exogenous hormones: first an auxin (2,4-D) then a cytokinin (kinetin). Our observations indicated that secondary somatic embryos could already develop in the proliferative calli derived from immature zygotic embryo tissues within one week from the start of in vitro culture. Cell states and tissue identity were deduced from detailed histological examination, and in situ hybridization was performed to map the expression of key developmental genes. The fast SE induction method we describe here facilitates the mechanistic study of the processes involved and may significantly shorten the production of transgenic or gene-edited plants.

Multiple Plant Regeneration from Embryogenic Calli of Paulownia tomentosa (Thunb.) Steud.

The aim of this paper was to study the effect of plant growth regulators on callus induction and in vitro morphogenesis using various explants of Paulownia tomentosa to develop an efficient plant regeneration protocol. Different plant organ sections (leaves, apical shoot tips, petals, nodes, and internodes) were cultured as explants to identify the best in vitro explants responsive to callus induction and plant regeneration. Explants were cultivated on MS media supplemented with different concentrations of plant growth regulators (TDZ (Thidiazuron), BAP (6-Benzylaminopurine), kinetin, and NAA (1-Naphthaleneacetic acid). It was discovered that the addition of TDZ and NAA stimulated the induction of somatic embryogenesis. It was discovered that the MS medium with the combination of plant growth regulators BAP (35.5 µM) and NAA (5.4 µM) with the addition of 30.0 g/L maltose, 500.0 mg/L casein hydrolysate, and 250.0 mg/L L-proline was optimal for callus induction and multiple plant regeneration. The study of the regenerative capacity of various explants of Paulownia tomentosa in vitro showed that plant regeneration depends on the type of explant, and occurs in both ways, indirectly, through the formation of callus tissues and directly on the explant, without callus formation. As a result of this study, the efficient reproducible protocol of embryogenic callus formation and multiple shoot induction in vitro of Paulownia tomentosa was developed. This system provides a clear increase in the frequency of plant regeneration from 36.3 ± 3.4% to 38.6 ± 2.3% per embryogenic callus from leaves and apical shoot tips, respectively.

MiR171 modulates induction of somatic embryogenesis in citrus callus.

Overexpression of miR171 restored SE competence in the recalcitrant citrus callus, and inhibition of miR171 function weakened SE competence in the strongly embryogenic citrus callus. Somatic embryogenesis (SE) is an important way of in vitro regeneration for plants. For perennial woody crops such as citrus, embryogenic callus is usually induced from unfertilized aborted ovules and widely used in biotechnology aided breeding. However, SE capacity always declines in callus during subculture, which makes regeneration difficult and hinders the application of biotechnology. We previously found that miR171 may be a regulator of SE in citrus, based on the abundant expression of csi-miR171c in the embryogenic callus and during SE of citrus. Here, we report that miR171 promotes SE and is required for SE in citrus. Overexpression of miR171 restored SE competence in the recalcitrant callus of 'Guoqing No.1' Satsuma mandarin (G1), whereas inhibition of miR171 function by Short Tandem Target Mimic (STTM) weakened SE competence in the strongly embryogenic callus of 'Valencia' sweet orange (V). The comparative transcriptomic analysis in miR171 overexpressed callus line (OE) and the wild type callus (WT) indicated that overexpression of miR171 decreased the expression level of its SCARECROW-LIKE (CsSCL) targets, and activated stress response related biological processes and metabolic processes that are required for cell differentiation. However, CsSCLs were up-regulated in the OE callus during SE induction process, which activated the cell division and developmental processes that are required for embryogenesis progress. Our results validate the function of miR171 in regulation of SE and reveal the biological responses provoked by miR171 in citrus that may promote SE.

VARIACIÓN SOMACLONAL EN PLANTAS DE Phalaenopsis sp. var. Dudú REGENERADAS POR EMBRIOGÉNESIS SOMÁTICA DIRECTA

<p><strong>Background</strong>: Nowadays, a growing demand for hybrids of <em>Phalaenopsis</em> sp. exists to satisfy this demand it is necessary to develop protocols for massive propagation that ensure high percentages of clonal regeneration, such as somatic embryogenesis. Besides, studying genetic variation within regenerated plants offers a greater understanding of the suitability of the micropropagation protocol in relation with genetic stability of the materials used. <strong>Objective</strong>: The present research work aimed to evaluate three concentrations of two types of plant growth regulators (RCV). 6-benzylaminopurine cytokinin (BA) (1.0, 2.0 y 3.0 mgL<sup>-1</sup>) in combination with three concentrations of 2,4-diclorofenoxiacetic acid (2,4-D) (3.0, 4.0, 5.0 mgL<sup>-1</sup>), for the induction of somatic embryos. In addition, the genetic stability of the regenerated plants was analyzed using molecular markers type RAPD (Random Amplified Polymorphic DNA). <strong>Methodology</strong>: The induction of somatic embryogenesis was induced from two leaf explants with different stages of develop from 15-20 cm in height <em>Phalaenopsis</em> sp. var. Dudú seedlings, cultivated <em>in vitro</em>; first leaf as mature explant (PH) and third leaf as young explant (TH). <strong>Results</strong>: The highest number of regenerated plants was 29.8 at 135 days after the start of the culture (ddic) with 2.0 and 5.0 mgL<sup>-1</sup> of BA and 2,4-D, respectively, using TH as explant. In the morphogenetic response of the regenerated explants, a correlation was observed between the age of the explant and the RCV concentration. Polymorphic bands were observed with the four primers used, indicating somaclonal variation in regenerated plants. <strong>Implications</strong>: The results obtained provide an alternative for regeneration, as well as offering a methodology to initiate genetic improvement programs in <em>Phalaenopsis</em> sp. var. Dudú. <strong>Conclusions</strong>: <em>In vitro</em> regeneration of <em>Phalaenopsis</em> sp. var. Dudú by somatic embryogenesis was achieved, as well as the analysis of the genetic integrity of the regenerated material. </p>

MiR160 Interacts in vivo With Pinus pinaster AUXIN RESPONSE FACTOR 18 Target Site and Negatively Regulates Its Expression During Conifer Somatic Embryo Development.

MicroRNAs (miRNAs) are key regulators of several plant developmental processes including embryogenesis. Most miRNA families are conserved across major groups of plant species, but their regulatory roles have been studied mainly in model species like Arabidopsis and other angiosperms. In gymnosperms, miRNA-dependent regulation has been less studied since functional approaches in these species are often difficult to establish. Given the fundamental roles of auxin signaling in somatic embryogenesis (SE) induction and embryo development, we investigated a previously predicted interaction between miR160 and a putative target encoding AUXIN RESPONSE FACTOR 18 in Pinus pinaster (PpARF18) embryonic tissues. Phylogenetic analysis of AUXIN RESPONSE FACTOR 18 (ARF18) from Pinus pinaster and Picea abies, used here as a model system of conifer embryogenesis, showed their close relatedness to AUXIN RESPONSE FACTOR (ARF) genes known to be targeted by miR160 in other species, including Arabidopsis ARF10 and ARF16. By using a luciferase (LUC) reporter system for miRNA activity in Arabidopsis protoplasts, we have confirmed that P. pinaster miR160 (ppi-miR160) interacts in vivo with PpARF18 target site. When the primary miR160 from P. pinaster was overexpressed in protoplasts under non-limiting levels of ARGONAUTE1, a significant increase of miR160 target cleavage activity was observed. In contrast, co-expression of the primary miRNA and the target mimic MIM160 led to a decrease of miR160 activity. Our results further support that this interaction is functional during consecutive stages of SE in the conifer model P. abies. Expression analyses conducted in five stages of development, from proembryogenic masses (PEMs) to the mature embryo, show that conifer ARF18 is negatively regulated by miR160 toward the fully developed mature embryo when miR160 reached its highest expression level. This study reports the first in vivo validation of a predicted target site of a conifer miRNA supporting the conservation of miR160 interaction with ARF targets in gymnosperms. The approach used here should be useful for future characterization of miRNA functions in conifer embryogenesis.

Constitutive Overexpression of a Conifer WOX2 Homolog Affects Somatic Embryo Development in Pinus pinaster and Promotes Somatic Embryogenesis and Organogenesis in Arabidopsis Seedlings.

Although full sequence data of several embryogenesis-related genes are available in conifers, their functions are still poorly understood. In this study, we focused on the transcription factor WUSCHEL-related HOMEOBOX 2 (WOX2), which is involved in determination of the apical domain during early embryogenesis, and is required for initiation of the stem cell program in the embryogenic shoot meristem of Arabidopsis. We studied the effects of constitutive overexpression of Pinus pinaster WOX2 (PpWOX2) by Agrobacterium-mediated transformation of P. pinaster somatic embryos and Arabidopsis seedlings. Overexpression of PpWOX2 during proliferation and maturation of somatic embryos of P. pinaster led to alterations in the quantity and quality of cotyledonary embryos. In addition, transgenic somatic seedlings of P. pinaster showed non-embryogenic callus formation in the region of roots and subsequently inhibited root growth. Overexpression of PpWOX2 in Arabidopsis promoted somatic embryogenesis and organogenesis in a part of the transgenic seedlings of the first and second generations. A concomitant increased expression of endogenous embryogenesis-related genes such as AtLEC1 was detected in transgenic plants of the first generation. Various plant phenotypes observed from single overexpressing transgenic lines of the second generation suggest some significant interactions between PpWOX2 and AtWOX2. As an explanation, functional redundancy in the WOX family is suggested for seed plants. Our results demonstrate that the constitutive high expression of PpWOX2 in Arabidopsis and P. pinaster affected embryogenesis-related traits. These findings further support some evolutionary conserved roles of this gene in embryo development of seed plants and have practical implications toward somatic embryogenesis induction in conifers.

MiR172 Regulates WUS during Somatic Embryogenesis in Arabidopsis via AP2.

In plants, the embryogenic transition of somatic cells requires the reprogramming of the cell transcriptome, which is under the control of genetic and epigenetic factors. Correspondingly, the extensive modulation of genes encoding transcription factors and miRNAs has been indicated as controlling the induction of somatic embryogenesis in Arabidopsis and other plants. Among the MIRNAs that have a differential expression during somatic embryogenesis, members of the MIRNA172 gene family have been identified, which implies a role of miR172 in controlling the embryogenic transition in Arabidopsis. In the present study, we found a disturbed expression of both MIRNA172 and candidate miR172-target genes, including AP2, TOE1, TOE2, TOE3, SMZ and SNZ, that negatively affected the embryogenic response of transgenic explants. Next, we examined the role of AP2 in the miR172-mediated mechanism that controls the embryogenic response. We found some evidence that by controlling AP2, miR172 might repress the WUS that has an important function in embryogenic induction. We showed that the mechanism of the miR172-AP2-controlled repression of WUS involves histone acetylation. We observed the upregulation of the WUS transcripts in an embryogenic culture that was overexpressing AP2 and treated with trichostatin A (TSA), which is an inhibitor of HDAC histone deacetylases. The increased expression of the WUS gene in the embryogenic culture of the hdac mutants further confirmed the role of histone acetylation in WUS control during somatic embryogenesis. A chromatin-immunoprecipitation analysis provided evidence about the contribution of HDA6/19-mediated histone deacetylation to AP2-controlled WUS repression during embryogenic induction. The upstream regulatory elements of the miR172-AP2-WUS pathway might involve the miR156-controlled SPL9/SPL10, which control the level of mature miR172 in an embryogenic culture.

Somatic embryogenesis: from discovery through investigation to application

Plant cells possess the remarkable ability to adapt to environmental changes. It is manifested by formation of embryos directly from the cells of plant body, bypassing the fertilization stage. These embryo structures develop into complete plants. The process itself, to distinguish the path of formation and emphasize consistency with zygotic embryogenesis, is referred to as somatic embryogenesis (SE). Although more than 60 years have passed since the first publication on the phenomenon has been written, the mechanism of reprogramming of a somatic cell into an embryogenic one is still not fully understood. This is a critical step in SE that can be induced by exo- and endogenous factors and stress treatments. The exposition of plant material to these factors affects the reorganization of the chromatin structure and gene expression, which can consequently trigger the program of embryogenesis. The paper reviews current knowledge on how the identity of totipotent cells is determined and the which stimuli are required to reprogram somatic cell development. Knowledge of key molecular regulators and the network of relationships that control the SE induction is summarized. Issues that are important for enhancing the understanding of the mechanisms underlying totipotency are also defined. Finally, the practical potential of SE is demonstrated, and examples of its use are provided.

Overexpression of Oil Palm Early Nodulin 93 Protein Gene (EgENOD93) Enhances In Vitro Shoot Regeneration in Arabidopsis thaliana.

EgENOD93 was first identified in a cDNA microarray study of oil palm tissue culture where it was highly expressed in leaf explants with embryogenic potential. Functional characterization via an RNA interference study of its orthologue in Medicago truncatula demonstrated a significant role of this gene in somatic embryo formation. In this study, EgENOD93 was overexpressed in the important model plant Arabidopsis thaliana to investigate the embryogenic potential of EgENOD93 transgenic Arabidopsis explants compared to explants from control plants (pMDC140 and WT). Experiments using leaf explants revealed higher numbers of regenerated shoots at day 27 in all the homozygous transgenic Arabidopsis cultures (Tg01, Tg02 and Tg03) compared to controls. The expression level of EgENOD93 in Arabidopsis cultures was quantified using reverse transcription quantitative real-time PCR (RT-qPCR). The results supported the overexpression of this gene in transgenic Arabidopsis cultures, with 6 and 10 times higher expression of EgENOD93 in callus at Day 9 and Day 20, respectively. Overall, the results support the role of EgENOD93 in the enhancement of shoot regeneration in transgenic Arabidopsis. This together with the previous results observed in oil palm and Medicago truncatula suggests that ENOD93 plays a key role in the induction of somatic embryogenesis. A similarity to early nodulation-like ontogeny is possible.

Somatic embryogenesis from flower tepals of Hippeastrum aiming regeneration of virus-free plants

Hippeastrum hybridum is an important bulbous flower plant in world floriculture, which are propagated conventionally by the technique known as double or twin scales to obtain plants with clonal origin. However, this technique promotes the propagation of systemic diseases, particularly mosaic-inducing viruses. The aim of this paper was to evaluate the somatic embryogenesis (SE) from tepals as an alternative to provide a technique for SE induction and to obtaining virus-free plantlets of Hippeastrum from infected plants. The concentrations of 2,4-Dichlorofenoxiacetic Acid (2,4-D) and thidiazuron (TDZ) was evaluated in SE induction pathway. The monitoring of viruses during the assays with tepals was performed by Reverse Transcription-Polymerase Chain Reaction. SE induction was obtained, for the first time, in tepal segments from flower buds of Hippeastrum. The 2,4-D was the main factor for embryogenic callus induction, and TDZ increased the SE induction rate. However, conversion of somatic embryos into plantlets were only developed in free-2,4-D media, replaced by 1.0 mg L−1 6-Benziladenine. Out of five virus species monitored during the experiment, Cucumber mosaic virus was detected in tepals and Hippeastrum mosaic virus in leaves of donor plants. The SE-derived plantlets that germinated in vitro were acclimatized and tested negative for all viruses assayed.

Somatic Embryogenesis of Anthurium andraeanum Linden., -ATropical Florists'Plant.

The global floriculture market is expected to reach US$41.1 billion by 2027 at a CAGR of 5% over the analysis period 2020-2027; on the year 2020, the recorded market value in this trade was US$29.2 billion. The florists mainly useAnthurium andraeanum flowers in fashionable bouquets and floral arrangements because of their beautiful, attractive bright colored eye-catching spathe, candle-like spadix, prolonged vase life, etc. The cut flower industry always seeks elite cultivars and new hybrids of A. andraeanum, that in turn depend on the availability of large numbers of clonal planting propagules. In vitro somatic embryogenesis is an important technique for large-scale clonal propagation, development of transgenic plants, creation of new variety by somaclonal variation, mutagenesis on in vitro plants, and germplasm preservation for future use. Here, we describe the protocol of somatic embryogenesis of Anthurium andraeanum cv. Cancan, an important commercial cultivated variety. The protocol has been optimized by using 4 different types of culture mediawhich are used during embryogenic callus induction, multiplication of callus, induction of somatic embryogenesis, and maturation plus conversionof embryosinto plantlets. The protocol outlines the detailed methods from mother plant procurement to hardening of micro plants that is ready to transfer in the field and itcan be used for large-scale commercial propagation.

Efficient Rebalancing of ROS Levels in Plants Links to Temporarily Enhanced Aerobic Fermentation, Distinct Cell Restructuration, and Resilience in Field

Efficient Rebalancing of ROS Levels in Plants Links to Temporarily Enhanced Aerobic Fermentation, Distinct Cell Restructuration, and Resilience in Field

Use of Thin Cell Layer (TCL) to Obtain Somatic Embryogenesis.

The thin cell layer (TCL) culture system was initially reported in relation to the model plant Nicotiana tabacum, giving rise to 47years of continuous application and investigation on micropropagation and plant breeding of over 100 plant species or hybrids. The small sizes of the tissue sections (100μm to 1-2mm in thickness), its classification into transverse TCL (tTCL) or longitudinal TCL (lTCL) categories, and the interaction between the cultured cells and the culture medium are the main drivers of its efficacy in tens of plants for the induction of somatic embryogenesis, relative to the conventional in-vitro culture system. Furthermore, it promotes higher productivity and reduced time in the proliferation of cultures, which is key for the differentiation of cells and plant tissues. This chapter describes the main characteristics of the TCL sections, and the interaction between cells under in-vitro culture. In addition, it highlights the latest findings reporting the success of TCL in ornamental, herbaceous, woody, and recalcitrant plants. In most cases, studies on the use of TCL in combination with techniques such as bioreactors, histology, genetic transformation, and fidelity analysis, provide indisputable evidence that highlights the importance of this technique in plant biotechnology. Finally, the perspectives on TCL use are described, underlining the advantages and constraints of the technique for its continued use and future application.

Use of Thin Cell Layer Technique for Induction of Somatic Embryogenesis of Agave fourcroydes.

Agave fourcroydes (henequén) is a plant used for the extraction of hard fiber from its leaves. Due to its long-life cycle, it is very difficult to genetically improve. Somatic embryogenesis (SE) is a very useful micropropagation technique, that can be used for genetic improvement programs and increase the micropropagation of this species. SE is a morphogenic process by which somatic embryos are generated from somatic cells reprogramming. To initiate the regeneration program, the loss of cell-cell communication is suggested to be important. The Thin Cell Layer (TCL) technique allows for the isolation of specific cell or tissue layers, and in conjunction with strictly controlled growth conditions, may lead to the in vitro induction of specific morphogenic programs. Here, we describe a new protocol for the induction of somatic embryogenesis through TCL culture technique, from stem of elite clonal A. fourcroydes vitroplants previously generated through micropropagation of adventitious shoots.

RNAi Expression in Cotton for Control of Herbivorous Insects.

Cultivated cotton (Gossypium hirsutum) is heavily attacked by various species of insects worldwide and breeding of new varieties resistant to pests is still a hard battle to win. RNAi technology is an important reverse genetics tool to induce gene silencing in eukaryotic organisms and produce phenotypic modifications. In cotton, RNAi was applied to investigate gene function and enhance resistance to insects and pathogens. Different methods and techniques can be used to synthetize double stranded RNA (dsRNA) into plant cells. The Agrobacterium-mediated transformation is a common method to introduce RNAi binary plasmids into cotton genome and obtain stable transgenics plants. This methodology includes the coculture of cotton tissues with Agrobacterium cultures, selection of transgenic cells and induction of somatic embryogenesis to finally obtain transgenic plants after a relatively long period of time. The transient synthesis of dsRNA mediated by virus-induced gene silencing (VIGS) in cotton is an alternative to anticipate the silencing effect of a specific RNA sequence, prior to the development of a stable transgenic plant. VIGS vectors are incorporated into the plant by agroinfiltration technique. During VIGS replication inside plant cells, synthetized dsRNA allows the study on specific heterologous gene expression including the phenotypic effect on herbivorous target pests, thus facilitating a rapid evaluation of dsRNA expressed in cotton plants against individual insect target genes. Here we describe the complementation of these two techniques to evaluate RNAi-based cotton plant protection against insect pests.

Callogenesis and somatic embryogenesis of <i>Oryza sativa</i> L. (cv. MARDI Siraj 297) under the influence of 2, 4-dichlorophenoxyacetic acid and kinetin

<abstract> <p>Callogenesis and embryogenesis are integral parts of many tissue culture procedures for genetic manipulation in rice. However, the efficiency of both processes is largely dependent on the media constituent especially the plant growth regulators (PGRs) due to the genotype-dependent nature of <italic>in vitro</italic> culture protocols. Therefore, this study investigates the effect of two PGRs; 2, 4-dichlorophenoxyacetic acid (2, 4-D) and kinetin (Kin) on callus growth and somatic embryogenesis of an important Malaysian rice cultivar (<italic>Oryza sativa</italic> L. cv. MARDI Siraj 297). Mature rice seeds explants were inoculated in Murashige & Skoog (MS) medium supplemented with different combinations of 2, 4-D (0 to 3.5 mg/L) and Kin (0 to 0.5 mg/L) to induce callogenesis. Parameters for callus growth such as fresh weight (FW), callus induction frequency (CIF), embryogenic callus frequency (ECF), regeneration frequency (RF), number of plantlets per callus (PPC), callus texture and callus color were observed after 35 days of inoculation. The results show that the maximum callus growth was achieved in MS medium supplemented with combination of 2.0 mg/L 2, 4-D and 0.2 mg/L Kin, represented by the highest FW (211 mg), CIF (95%), ECF (90%), RF (100%) and PPC (22 plantlets); along with friable callus texture. Low concentration of 2, 4-D (0 to 0.5 mg/L) in the presence or absence of Kin promotes root growth instead of callus, while high concentrations (above 3.0 mg/L) retard the callus formation. The embryogenic calli from this optimized PGRs combination were successfully formed shoots in MS medium supplemented with 2 mg/L BAP and 1 mg/L NAA, followed by rooting in PGRs-free MS medium. This finding provides an efficient protocol for callogenesis and somatic embryogenesis of MARDI Siraj 297, since this is the first published report regarding somatic embryogenesis induction of this cultivar.</p> </abstract>