Mature Explants Research Articles

Severe acute liver disease in adults: Contemporary role of histopathology.

Liver biopsies have consistently contributed to our understanding of the pathogenesis and aetiologies of acute liver disease. As other diagnostic modalities have been developed and refined, the role of biopsy in the management of patients with acute liver failure (ALF), acute-on-chronic liver failure (ACLF) and acute hepatitis, including acute liver injury (ALI), has changed. Liver biopsy remains particularly valuable when first-line diagnostic algorithms fail to determine aetiology. Despite not being identified as a mandatory diagnostic tool in recent clinical guidelines for the management of ALF or ACLF, many centres continue to undertake biopsies given the relative safety of transjugular biopsy in this setting. Several studies have demonstrated that liver biopsy can provide prognostic information, particularly in the context of so-called indeterminate hepatitis, and is extremely useful in excluding conditions such as metastatic tumours that would preclude transplantation. In addition, its widespread use of percutaneous biopsies in cases of less severe acute liver injury, for example in the establishment of a diagnosis of acute presentation of autoimmune hepatitis or confirmation of a probable or definite drug-induced liver injury (DILI), has meant that many centres have seen a shift in the ratio of specimens they are receiving from patients with chronic to acute liver disease. Histopathologists therefore need to be equipped to deal with these challenging specimens. This overview provides an insight into the contemporary role of biopsies (as well as explant and autopsy material) in diagnosing acute liver disease. It outlines up-to-date clinical definitions of liver injury and considers recent recommendations for the diagnosis of AIH and drug-induced, autoimmune-like hepatitis (DI-AIH).

Application of mannitol as pre-treatment and sucrose supports callus induction through anther culture in Gossypium arboreum

Diploid or tree cotton (Gossypium arboreum) species may provide a breeding source for fiber quality and disease resistance. Developing homozygous lines of Gossypium arboreum through anther culture can be a successful strategy for breeding the parental lines needed for cotton hybrids. This study describes callus induction on four G. arboreum genoytypes under various concentrations of sucrose as well as plant growth regulators. Explant materials were derived from immature anther and grew on modified Murashige and Skoog (MS) media. Prior to main treatment, mannitol application were applied to anthers with concentration of 0.7 M and modified MS media consisted of 3%, 5%, and 7% sucrose, as well as an addition of IAA, NAA, BAP, 2,4-D and Kin. Optimum callus formation was observed in Marvi genotype, in a medium containg IAA as well as sucrose between 5-7%. Furthermore, combination of IAA and NAA with sucrose 7% indicates highest number of embryogenic calli. However, Marvi genotype showed decreased rate of callus formation when the amount of sucrose increased on MS supplemented with NAA medium. The results indicated that the best formula on each genotypes were different. However, MS media with IAA (1 mg/l) + kin (0.2 mg/l) + CM (70 ml/l) + sucrose 5% provided the best performance to produce embryogenic calli on each genotypes. Our report can be utilized as an opportunity and strategy to obtain double haploid lines as a parental source in hybrid production.

Tissue Culture Response and In Vitro Plant Regeneration of Malus 'Baiyun' (a New Cultivar of Ornamental Crabapple).

Malus 'Baiyun' (registration no. 20210210), a new crabapple cultivar, was registered in 2021 by the Nanjing Forestry Unversity. However, the difficult rooting has greatly limited the production of high-quality M. 'Baiyun' in industrialization development. There is thus a pressing need to develop an organogenesis protocol for the in vitro propagation of M. 'Baiyun' to alleviate a shortage of high-quality M. 'Baiyun' seedlings. The results showed that choosing the apical bud in mid-March was an excellent explant material. To promote proliferation, the highest proliferation (6.27) of apical shoots was cultured on Murashige and Skoog (MS) medium supplemented with 0.5 mg·L-1 6-benzylaminopurine(6-BA) + 0.05 mg·L-1 indole-3-butyric acid (IBA). Subsequently, a 100% rooting rate, average number of roots per shoot of 6.2 and maximum length of roots of 4.96 cm were obtained on half-strength Murashige and Skoog (1/2 MS) medium with the application of 0.5 mg·L-1 naphthaleneacetic acid (NAA) or 0.6 mg·L-1 NAA + 0.7 mg·L-1 IBA. Additionally, thick and lateral roots were obtained with 0.6 mg·L-1 NAA + 0.7 mg·L-1 IBA. Our study is the first to establish an effective organogenesis protocol for new crabapple cultivars using stem segments.

Improvement of <i>in vitro</i> shoot regeneration and flowering from petal explants of <i>Begonia X hiemalis</i> fotsch

Begonias hold significant importance due to their use as culinary plants, medicinal remedies, decorative elements, and high-value crops. This research aims to find new explant materials for regeneration shoots and direct flowering with high frequency. In this study, high-frequency shoot regeneration and in vitro flowering from petal explants of Begonia x hiemalis Fotsch were achieved. Whole petal explants disinfected with an AgNPs solution at 0.5 g/L for 15 min achieved the highest survival rate. The highest shoot regeneration rate reached 86.66%, with 65.33 shoots/explant from ½-petal explants. Direct formation of the flower consists of two different structural forms, including single petal clusters and complete flower buds, with the corresponding flower formation rates of 19.66 petals/explant; 0.53 flower buds/explant, respectively, at the keel of the petals on the MS medium supplemented with 1000 mg/L myo-inositol, 1.0 mg/L BA, 1.0 mg/L NAA, 30 g/L sucrose and 9.0 g/L agar. Interestingly, increasing the concentration of myo-inositol in the culture medium led to the appearance of red-leaf shoots at the highest rate (12.66 shoots/explant), reaching a maximum shoot height of 1.40 cm after 8 weeks of culture. After each subculture, the red color on leaves tended to decrease gradually, and they were in vitro flowered after the second subculture at a rate of 15% and the third subculture at a rate of 10% on medium supplemented with 40 mg/L adenine, 1.0 mg/L NAA, 1.0 mg/L BA, 30 g/L sucrose, 9 g/L agar and 1 g/L activated charcoal. The plantlets regenerated from petal cultures bloomed with standard red pigments, similar to those grown in natural growing conditions.

Correlating microstructure and mechanical properties of harvested high dose Zorita light water reactor internals

In this study, microstructural studies and micro-mechanical testing of an ex-plant material harvested from the decommissioned pressurized water reactor (PWR) is carried out. Irradiated 304 stainless steel (SS) components were harvested by the Electric Power Research Institute, U.S. Nuclear Regulatory Commission, and members of the José Cabrera Nuclear Power Station (Zorita) in Spain. The bi-crystalline micro-tensile specimens, atom probe tomography (APT) tips, and transmission electron microscopy (TEM) lamellae were fabricated from the baffle plate materials irradiated to 0.05, 15 and 50 dpa to elucidate microstructural, microchemical, and local mechanical properties changes as a function of dose. TEM and APT studies reveal radiation induced Ni, Cr, and Si rich precipitates in the 15 dpa sample that become smaller and partially redissolve into the matrix of the 50 dpa sample (decreasing size and number density). Dislocation loop and cavity number density and size were quantified as a function of dose as well as swelling. Intergranular Ni and Si enrichments with concomitant Fe and Cr depletion were observed in the 15 dpa sample and were more pronounced in the 50 dpa sample. Micro-tensile testing performed in the scanning electron microscope (SEM) at room temperature and 300 °C shows that the material's local yield strength and ultimate tensile strength decreases with increased dose and elevated test temperature and provides further insights via localized strain mapping. Current and previous mechanical data was compared with calculated values using the dispersed barrier hardening model with inputs of dislocation loops, precipitates, and cavities from microstructural characterization.

Modulating ascorbic acid levels to optimize somatic embryogenesis in Picea abies (L.) H. Karst. Insights into oxidative stress and endogenous phytohormones regulation.

Global warming has adversely affected Picea abies (L.) H. Karst. forests in Europe, prompting the need for innovative forest-breeding strategies. Somatic embryogenesis (SE) offers promise but requires protocol refinement. Understanding the molecular mechanisms governing somatic embryo development is essential, as oxidative stress plays a crucial role in SE regulation. Ascorbic acid (ASA), is a vital antioxidant that can potentially control oxidative stress. In the present study, we normalized ASA concentrations in induction and proliferation media to enhance embryogenic tissue (ET) regeneration and proliferation capacity of mature explants. The media were supplemented with ASA at 0 mg l-1, 25 mg l-1, 50 mg l-1, 100 mg l-1, and 200 mg l-1. The accumulation of hydrogen peroxide (H2O2) and endogenous phytohormones, including auxins, cytokinins, brassinosteroids, abscisic acid, and gibberellin, was measured in non-embryonic calli and ET. Subsequently, their impact on ET induction and multiplication was analyzed. Our results demonstrate that application of ASA at concentrations of 25 mg l-1 and 200 mg l-1 led to increased H2O2 levels, potentially inducing oxidative stress while simultaneously reducing the levels of all endohormone groups. Notably, the highest ET induction frequency (approximately 70%) was observed for ASA at 50 mg l-1. These findings will enhance SE induction procedures, particularly in more resistant explants, underscoring the significance of ASA application to culture media.

Rapid Establishment of a Regeneration System for Astragalus membranaceus Mongholicus

Background: Astragalus membranaceus Mongholicus (Astragalus) is one of the most widely used medicinal plants in the world. It is a traditional Chinese medicine plant that has been used to treat various human diseases and conditions, such as inflammation, gastrointestinal bacterial infections and malignant tumors. With the expansion of artificial planting areas, the demand for high-quality Astragalus seedlings is also increasing. The traditional seedling-raising methods of direct seeding and induced callus have problems such as a long growth period, unstable genetic traits, strict sampling and rooting difficulties. Therefore, it is very important to multiply the excellent germplasm by optimizing the tissue culture regeneration system. Methods: In this study, Astragalus field cultivated plants, which are easily available, were used as materials and the method of tissue culture cuttings was used to select the direct inoculation method of stem segments as explant materials to establish a plant regeneration system of Astragalus and the best sampling period, sampling site and optimal medium for regeneration of field cultivated plants were screened. Result: The best sampling period for plant regeneration of Astragalus in the field was about 40 d after the emergence of the 3-year-old Astragalus and the rooting rate of the regenerated plants was 100.00%. The best sampling site is the middle section of the stem and the regeneration system using the middle section of the stem as the explant is better than using the top and bottom of the stem as the explant and the rooting rate of the regenerated plants is 100.00%; the best medium for the regenerated plants is N22: 1/2MS+1 mg/L NAA and T28: 1/2MS+1 mg/L ABT, the rooting rates were 76.67% and 96.67%, respectively. The regenerated plants established with the stems of the field cultivated plants of Astragalus as explants were all able to induce seedlings after 1 week and the regenerated seedlings start to take root after 3 weeks of growth and can be transplanted and propagated after 6 weeks of culture. The regeneration system established in this study by direct cuttings from cultivated Astragalus stems is more rapid and efficient, breaking through the difficulties of limited access to material and long growth period of regenerated seedlings in the traditional regeneration system and providing technical support for the conservation and utilization of wild resources of Astragalus and rapid expansion of new varieties and germplasm conservation.

Results of a monocentric field study: value of histology compared to sonication method and conventional tissue culture in the diagnosis of periprosthetic joint infection (PJI).

To confirm the diagnosis of periprosthetic joint infection (PJI), the Infectious Diseases Society of America (IDSA) and the International Consensus Meeting (ICM) have defined criteria that include histology as a minor criterion and the sonication method only as an additional criterion. The aim of this monocentric, retrospective study was to investigate the value of histology and whether sonication leads to a more accurate diagnosis. All revision surgeries for knee and hip arthroplasty between 2017 and 2020 were included. With regard to microbiological diagnostic, conventional culture of periprosthetic biopsies and sonication of explant material were performed. In addition, histology and non-specific inflammatory markers (CRP, leukocytes) were recorded. A total of 78 patients with PJI and 62 aseptic controls were included. From both microbiological methods (conventional culture / sonication), Staphyloccus (S.) epidermidis and S. aureus were detected most frequently. However, compared to the conventional microbiology, a higher sensitivity was calculated for sonication, albeit with a lower specificity in relation to a PJI. In two logistic regression models for the significance of all diagnostic parameters in PJI, the AUC was 0.92 and 0.96 with histology in particular making the decisive contribution in both models (p < 0. 001, both models). Since histology showed the highest accuracy in the current study, its importance in the PJI criteria should be reevaluated. Sonication shows a high sensitivity for germ detection with a lower specificity and should only be used in combination with the conventional culture for microbiolgical diagnostics.

Protocol for efficient ginseng transformation

Panax ginseng is a major medicinal crop with pharmaceutical efficacy derived from ginsenoside metabolites. Despite its genome information, the inefficiency of ginseng transformation hinders the study of the molecular mechanism of ginseng plant metabolism. Thus, this protocol aimed to develop an easy and efficient system for ginseng transformation. We established a transformation system using ginseng callus cultured in a liquid medium, which has a higher rate compared with cotyledon explant. In addition, Agrobacterium tumefaciens has been used for plant transformation. Compared with the LBA4404 strain, C58C1 was inappropriate for ginseng transformation using ginseng callus. We induced and maintained calli in liquid medium and cut them into small pieces before infection. After infection, we selected calli that survived from the selection media until identification of newly growing cells. In β-glucuronidase (GUS) assay, the expression of the GUS gene was observed in cells that were newly generated from explants. We treated calli with 0.05 M of MgSO4 before infection. After MgSO4 pre-treatment, the transformation efficiency of growing cells around infected callus was increased than non-treated control. Moreover, we constructed and introduced a visible reporter RUBY system to easily identify transformed cells. Using this system, we could identify the cells by a red color with naked eyes. Based on our transformation protocol, the success rate has increased to 77.27% in surviving lines during selection culture. This stable ginseng transformation could facilitate the overexpression and knockout of ginseng lines for functional or synthetic biological studies. We re-designed the ginseng transformation protocol using ginseng calli for explant material, and MgSO4 pretreatment was conducted before Agrobacterium infection. RUBY reporter was successfully introduced in ginseng.

In vitro callus and shoot initiation of Black cohosh (Actaea racemosa. L) pre-incubated in darkness treatments

A medicinal plant that is commonly overharvested in its native habitat, Black cohosh (Actaea racemosa L.) rhizomes are used to mitigate symptoms of menopause and are a common alternative to hormone replacement therapy. Developing an accurate protocol for initiation of Black cohosh in tissue culture is becoming a necessity. Wild harvesting practices are the main source of Black cohosh rhizomes processed by pharmaceutical companies and are causing a significant decrease of these wild plants, leading to an At-Risk status. Growing these plants in a plant tissue culture laboratory is more sustainable than current practices, and having an accurate protocol for initiation of these plants is crucial to this. Our lab has previously developed a protocol for initiation of Black cohosh and this research was meant to optimize this protocol. Leaf explants of Black cohosh were cultured in vitro to determine the optimal duration of time for initiation of callus and/or shoot under darkness, and appropriate plant growth regulators (PGR) for shoot growth. Leaf explants incubated under darkness for increasing amount of time (0 to 6 weeks of darkness) were analyzed at two stages of tissue development. Stage 1 involved initiation of leaf explant material under darkness (treatment) and measured the callus development in leaf explants; Stage 2 was induction of adventitious shoot growth and cell division and compared the effect of two PGRs, KT and BA, on shoot growth for each darkness treatment. Leaf explants were given a score using a culture chart that assigns a numerical score to leaf explants based on their physiological characteristics. These numerical scores took into account color of leaf explant, callus development, shoot growth, and presence of dead leaf tissue. Stage 2 measured the number of shoots produced per explant within culture containers between PGRs and the number of weeks in darkness. Overall trends indicated that mortality of leaf explants decreased as the duration of incubation under darkness increased with mortality of explants decreasing from 63.9% (T0 darkness treatment) to 2.8% (T6 darkness treatment). Culture scores of explants (including dead explants) was highest for T6 weeks of darkness (culture score 2.96 on average), and for culture scores without dead explants (culture score of 3.05). During the adventitious shoot growth stage, however, T5 weeks of darkness performed significantly higher (KT: 28 shoots/ BA: 17.3 shoots) than T6 weeks of darkness (KT: 12.9 shoots/ BA: 9.4) in Replication 2 for both plant growth regulators KT PGR performed better, on average, than BA PGR

Micropropagation of Arisaema spp. (filiforme and brinchangense): Explant selection and surface sterilization insights

Arisaema filiforme and A. brichangense are a perennial herbaceous plant (family Araceae) found distributed in mossy forest, Cameron Highlands, with the elevation of 1,900 meters above sea level (a.s.l). The unique inflorescence formation resembling cobra has given this plant the name Cobra lilies, and suitable to be planted as ornamental plant. In addition, it has been used traditionally as a herb. However, the population of these two species are very limited, only thrive in higher elevation and also considered as an endangered. Therefore, realizing its potential in the future as one of the new ornamental plant and materials for the herb bioindustry, a micropropagation approach was employed to produce these species in mass production. Seeds, rhizomes, and petioles were used as the explant materials, cultured onto Murashige and Skoog (MS) media supplemented with different concentrations (0, 0.5, 1, 2 mg L-1) of 6-Benzylaminopurine (BAP). The findings revealed rhizomes and seeds to be significant explants for micropropagation, where the survival rate for these two are more than 80%. Petioles had 0% of survivability after week eight of culture due to the fungi infection and tissue necrosis. This study provides an insight into explant selection, where different plant organs have different survival rate due to the tissue mechanical strength. Also, optimum surface sterilization process is very critical in micropropagation to avoid the contamination of the culture and also necrotizing.

Appropriate Condition for Cluster Buds Induction of &lt;i&gt;Cremastra appendiculata&lt;/i&gt;

To screen for suitable conditions for cluster bud induction of Cremastra appendiculata .&nbsp;The aseptic germination protocorms&nbsp;of C. appendiculata &nbsp;seeds were used as explant materials to explore the effects of different basic media, types of plant growth regulators, anti-browning agents&nbsp;and their concentrations&nbsp;on the induction of cluster buds&nbsp;of C. appendiculata .&nbsp;The results showed that: 1/4MS medium was the suitable basic medium for cluster buds growing; when the concentration of plant growth regulator TDZ was 2.0 mg/L and the concentration of IAA was 0.2 mg/L, the induction rates of cluster bud were higher which are 72.58% and 55.63% respectively; when the anti-browning agent was PVP and its concentration is 2&nbsp;000 mg/L, the induction rate of cluster buds was higher which is 79.90%. The suitable conditions for C. appendiculata cluster buds induction are: 1/4MS + TDZ 2.0 mg/L+ IAA 0.1 mg/L+ PVP 2&nbsp;000 mg/L, and the induction rate can reach 81.16%. This&nbsp;provided&nbsp;a basis for establishing a rapid propagation technology system of &nbsp;C. appendiculata &nbsp;and realizing its&nbsp;large-scale artificial cultivation.

An Investigation into Using Temporary Immersion Bioreactors to Micropropagate Moringa oleifera Lam. Callus, Roots, and Shoots

Moringa oleifera Lam., a tree naturally grown in the tropics, is becoming increasingly popular as an industrial crop due to its multitude of useful attributes. Therefore, this study tested the effect of temporary immersion system (TIS) bioreactors for mass micropropagation of Moringa oleifera Lam. callus, roots, and shoots. TIS are tissue culture systems that make use of timers to periodically immerse and drain plant cultures in a liquid nutrient medium instead of using solidified media. In initial studies, Moringa oleifera seeds were germinated in vitro, and in vitro seedling leaflets were then used as explant material for callus production on the pre-culturing media. Two experiments were conducted to improve the protocol for TIS bioreactor production. The first experiment investigated the effect of 6-benzylaminopurine (BA) and kinetin, whereas experiment 2 was conducted to improve shooting production. For the first experiment, leaf material was cultured onto a solidified medium consisting of half-strength Murashige and Skoog (MS) basal salts and 0.5 ppm 1-naphthaleneacetic acid (NAA) to initiate callus production before splitting it between solidified media and bioreactors for shooting. The shooting media consisted of full-strength MS basal salts and different treatments of kinetin and BA. A significant increase in callus production was observed with the use of TIS bioreactors, compared to solidified media, whereas root production had a highly significant interaction effect between the media and the cytokinin treatments. With shoot proliferation in mind, experiment 2 was performed, where microcuttings from in vitro-grown seedlings were excised and cultured onto a solidified MS medium, consisting of a control (0 ppm BA) and two different concentrations of 6-benzylaminopurine (BA) (0.1 ppm BA and 0.2 ppm BA) in the pre-culturing phase. Microcuttings were again excised after two weeks and transferred to the shooting media containing 0.1 ppm BA in TIS bioreactors and semi-solidified medium. Results showed TIS bioreactors to be effective in increasing both the amount and length of shoots produced. Shoot and callus fresh weights were also higher in explants cultured in TIS bioreactors. The results of this study also suggest M. oleifera sensitivities to plant growth regulators (PGRs). In conclusion, this study successfully produced callus, roots, and shoots in both the solidified media and TIS bioreactors, emphasizing the prospect of using TIS bioreactors for mass micropropagation of M. oleifera callus, roots, and shoots.

Impact of silver nitrate on the survival and regeneration potential of explants of Hippophae salicifolia (Seabuckthorn) D. Don

Hippophae salicifolia D. Don is an important plant of manifold benefits ranges from ecological refurbishment to socioeconomic development in their growing provinces. In vitro propagation practices in H. salicifolia hindered by various obstacles, among them explant necrosis or mortality is most important, especially when mature plants are the source for explants and in that case ethylene production by explants was supposed to be the main culprit. In this study, full strength Woody Plan medium with various hormonal combinations fortified with different silver nitrate/ AgNO3 concentrations of 0.5, 1.0, 1.5, 2.0 mg/L utilized to assess the impact of AgNO3 on survival and other developmental parameters in H. salicifolia mature and seedling explants. Silver nitrate efficiently controlled necrosis and reduced the explant mortality in the culture by enhancing survival percentages of the explants to the 80 ± 1.00 % in mature explants and 96 ± 0.45 %in seedling explants with supplementation of 2 mg/L in hormonal combination i.e., 15.0 mg/L BAP + 5.0 mg/L IAA + 2.0 mg/L GA3. Silver nitrate significantly influenced the development of average shoot numbers in seedling explants with highest average shoot number 53.4 ± 2.70 at 2 mg/L AgNO3 supplementation with hormonal combination 15.0 mg/L BAP + 5.0 mg/L IAA + 2.0 mg/L GA3, although shoot regeneration from mature explant remained modest to AgNO3 supplementations. In vitro generated shoots grown in media supplemented with silver nitrate when subjected for rooting, they failed to induce roots in rooting media.

Antioxidants Application Enhances Regeneration and Conversion of Date Palm (Phoenix dactylifera L.) Somatic Embryos.

Many embryogenic systems have been designed to generate somatic embryos (SEs) with the morphology, biochemistry, and vigor uniformity of zygotic embryos (ZEs). During the current investigation, several antioxidants were added to the maturation media of the developing somatic embryos of date palm. Explant material was a friable embryogenic callus that was placed in maturation media containing ABA at 0.5 mg L−1, 5 g L−1 polyethylene glycol, and 10 g L−1 phytagel. Furthermore, α-tocopherol or reduced glutathione (GSH) were used separately at (25 and 50 mg L−1). These treatments were compared to a widely used date palm combination of reduced ascorbic acid (ASC) and citric acid at 150 and 100 mg L−1, respectively, and to the medium free from any antioxidants. The relative growth percentage of embryogenic callus (EC), globularization degree, differentiation%, and SEs number were significantly increased with GSH (50 mg L−1). Additionally, the latter treatment significantly enhanced the conversion% of SEs and the number of secondary somatic embryos (SSEs). ASC and citric acid treatment increased leaf length, while α-tochopherol (50 mg L−1) elevated the number of leaves plantlet−1. GSH at 50 mg L−1 catalyzed the activities of polyphenol oxidase (PPO) and peroxidase (POD) in EC and enhanced the accumulation of proteins in SEs.

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

&lt;p&gt;&lt;strong&gt;Background&lt;/strong&gt;: Nowadays, a growing demand for hybrids of &lt;em&gt;Phalaenopsis&lt;/em&gt; 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. &lt;strong&gt;Objective&lt;/strong&gt;: 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&lt;sup&gt;-1&lt;/sup&gt;) in combination with three concentrations of 2,4-diclorofenoxiacetic acid (2,4-D) (3.0, 4.0, 5.0 mgL&lt;sup&gt;-1&lt;/sup&gt;), 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). &lt;strong&gt;Methodology&lt;/strong&gt;: The induction of somatic embryogenesis was induced from two leaf explants with different stages of develop from 15-20 cm in height &lt;em&gt;Phalaenopsis&lt;/em&gt; sp. var. Dudú seedlings, cultivated &lt;em&gt;in vitro&lt;/em&gt;; first leaf as mature explant (PH) and third leaf as young explant (TH). &lt;strong&gt;Results&lt;/strong&gt;: 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&lt;sup&gt;-1&lt;/sup&gt; 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. &lt;strong&gt;Implications&lt;/strong&gt;: The results obtained provide an alternative for regeneration, as well as offering a methodology to initiate genetic improvement programs in &lt;em&gt;Phalaenopsis&lt;/em&gt; sp. var. Dudú. &lt;strong&gt;Conclusions&lt;/strong&gt;: &lt;em&gt;In vitro&lt;/em&gt; regeneration of &lt;em&gt;Phalaenopsis&lt;/em&gt; sp. var. Dudú by somatic embryogenesis was achieved, as well as the analysis of the genetic integrity of the regenerated material. &lt;/p&gt;

Effect of type of mature embryo explants and acetosyringone on agrobacterium-mediated transformation of moroccan durum wheat

Drought is one of the major constraints in durum wheat production in the Mediterranean Basin. In order to overcome this problem, the genetic transformation of durum wheat is one of the choices for improvement. However, the recalcitrance to Agrobacterium-mediated transformation in durum wheat (Triticum turgidum L.) is one of the factors limiting a successful genetic transformation. The aim of this study was to investigate the effect of explant type and acetosyringone concentration for the efficient Agrobacterium-mediated genetic transformation of three Moroccan durum wheat varieties (Amria, Chaoui, and Marouane). The mature embryos (intact, halved and pieces) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pTF101.1 containing drought tolerance gene HVA1 from barley, and a selectable marker phosphinothricin (PPT) resistance (bar) gene. The explants were inoculated with A. tumefaciens (cell density OD650 at 0.7) at four different concentrations of acetosyringone (0, 100, 200, and 400 µM). The results showed that embryogenic calli from mature embryos showed higher regeneration and transformation than mature embryo halves and pieces. The integration of the transgene was confirmed by PCR amplification using primers specific to the bar gene, 2x35S promoter, and HVA1 gene. The transformation efficiency ranging from 0.33% to 2.33% was obtained in Amira variety using embryogenic calli and acetosyringone concentrations of 200 and 400 µM. The integration, as well as inheritance of the transgene, was confirmed by PCR amplification in T0 and T1 generations. This is the first report describing a genetic transformation of Moroccan durum wheat varieties via Agrobacterium tumefaciens.

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

&lt;abstract&gt; &lt;p&gt;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 &lt;italic&gt;in vitro&lt;/italic&gt; 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 (&lt;italic&gt;Oryza sativa&lt;/italic&gt; L. cv. MARDI Siraj 297). Mature rice seeds explants were inoculated in Murashige &amp;amp; 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.&lt;/p&gt; &lt;/abstract&gt;

Optimization of Somatic Embryo Regeneration of &lt;i&gt;Larix olgensis&lt;/i&gt; Through Multiple Media

To establish a stable and efficient&nbsp; Larix olgensis&nbsp; somatic embryo regeneration system. In this study, immature zygotic embryos of&nbsp; Larix olgensis &nbsp;were used as explant materials for embryogenic callus induction, and embryogenic callus with good growth were selected to induce somatic embryo formation by liquid medium A, semi-solid medium B, and solid medium C. The callus induction medium is S medium+2,4-D 1.0 mg/L + 6-BA 0.5 mg/L + KT 0.3 mg/L, sucrose 20 g/L, agar 3 g/L; the subculture medium is S medium+2, 4-D 0.15 mg/L + 6-BA 0.05 mg/L + KT 0.05 mg/L, sucrose 30 g/L, agar 6 g/L. Mature medium A is S medium + PEG4000 100 g/L + ABA 15 mg/L + AgNO 3 &nbsp;10 mg/L, sucrose 45 g/L liquid medium. Mature medium B and C are mature medium A supplemented with 2 g/L and 10 g/L agar, respectively. The maturation time of this method is about 32 days, the synchronization rate is 78.0%, and the proportion of somatic embryos in normal morphology is 83.0%. Somatic embryo formation efficiency in normal morphology can reach 223.0 embryos/g embryogenic callus, somatic embryo germination rate can reach 47.2%. Compared with a single maturation medium, the maturation time is shortened, somatic embryo generation is more synchronized, and high somatic embryo yield and quality are improved. It provides a way for the commercialization of somatic embryo regeneration of&nbsp; Larix olgensis , brings great convenience to large-scale breeding and reproduction, physiological and biochemical research, and genetic modification, and has great practical application value.

Plant Regeneration via Somatic Embryogenesis in Larix principis-rupprechtii Mayr

Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr) is a native conifer in North China with great economic and ecological values. Somatic embryogenesis (SE) is a powerful tool for the mass clonal propagation in plants. In this study, we described a high-efficiency SE system via indirect pathways and investigated the effect of genotype, culture conditions and phytohormones on SE. Immature zygotic embryos (IZEs) of L. principis-rupprechtii Mayr were used as explant materials. In the induction stage, embryogenic tissues (ETs) were induced on mLV medium supplemented with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L−1 6-benzylaminopurine (6-BA). The initiation frequencies showed significant differences (p &lt; 0.05) among 20 genotypes of open-pollinated mother trees with the highest induction frequency reaching 30%. For tissue proliferation, proliferation in liquid medium was more efficient compared with proliferation in semi-solid medium, providing a multiplication rate of 3.12 in an 8-day subculture period. As a necessary exogenous plant growth regulator (PGR) for somatic embryo maturation in conifers, abscisic acid (ABA) was optimized at 16 mg L−1 in this system. Next, an orthogonal test on osmotic pressure factors showed 50 g L−1 sucrose, 7 g L−1 phytagel and 75 g L−1 polyethylene glycol (PEG) was the optimal combination for somatic embryo maturation in L. principis-rupprechtii Mayr. Moreover, the dispersion culture method provided a more efficient somatic embryo maturation, up to 545 per gram of fresh weight (FW). Finally, 2 g L−1 of active charcoal (AC) was found to increase the somatic embryo germination rate to 63.46%. The improved protocol of SE will serve as a foundation for establishing mass propagation and genetic transformation of L. principis-rupprechtii Mayr.