Somatic Embryogenesis and Agrobacterium-Mediated Gene Transfer Procedures in Chilean Temperate Japonica Rice Varieties for Precision Breeding.

Rice (Oryza sativa) varieties are generated through breeding programs focused on local requirements. In Chile, the southernmost rice producer, rice productivity relies on the use and generation of temperate japonica germplasms, which need to be adapted to the intensifying effects of climate change. Advanced biotechnological tools can contribute to these breeding programs; new technologies associated with precision breeding, including gene editing, rely on procedures such as regeneration and gene transfer. In this study, the local rice varieties Platino, Cuarzo, Esmeralda, and Zafiro were evaluated for somatic embryogenesis potential using a process that involved the combined use of auxins and cytokinins. An auxin-based (2,4-D) general medium (2N6) allowed for the induction of embryogenic masses in all the genotypes. After induction, masses required culturing either in N6R (kinetin; Platino) or N6RN (BAP, kinetin, IBA, and 2,4-D; Cuarzo, Esmeralda, and Zafiro) to yield whole plants using regeneration medium (N6F, no hormone). The sprouting rates indicated Platino as the most responsive genotype; for this reason, this variety was evaluated for gene transfer. Fifteen-day-old embryo masses were assayed for Agrobacterium-mediated transformation using the bacterial strain EHA105 harboring pFLC-Myb/HPT/GFP, a modified T-DNA vector harboring a geminivirus-derived replicon. The vector included the green fluorescent protein reporter gene, allowing for continuous traceability. Reporter mRNA was produced as early as 3 d after agroinfiltration, and stable expression of the protein was observed along the complete process. These achievements enable further biotechnological steps in these and other genotypes from our breeding program.

Cross-species transmission and PB2 mammalian adaptations of highly pathogenic avian influenza A/H5N1 viruses in Chile.

H5N1 highly pathogenic avian influenza viruses (HPAIV) emerged in wild birds in Chile in December 2022 and spilled over into poultry, marine mammals, and one human. Between December 9, 2022 - March 14, 2023, a coordinated government/academic response detected HPAIV by real-time RT-PCR in 8.5% (412/4735) of samples from 23 avian and 3 mammal orders. Whole-genome sequences obtained from 77 birds and 8 marine mammals revealed that all Chilean H5N1 viruses belong to lineage 2.3.4.4b and cluster monophyletically with viruses from Peru, indicating a single introduction from North America into Peru/Chile. Mammalian adaptations were identified in the PB2 segment: D701N in two sea lions, one human, and one shorebird, and Q591K in the human and one sea lion. Minor variant analysis revealed that D701N was present in 52.9 - 70.9% of sequence reads, indicating the presence of both genotypes within hosts. Further surveillance of spillover events is warranted to assess the emergence and potential onward transmission of mammalian adapted H5N1 HPAIV in South America.

A Traceable DNA-Replicon Derived Vector to Speed Up Gene Editing in Potato: Interrupting Genes Related to Undesirable Postharvest Tuber Traits as an Example.

In potato (Solanum tuberosum L.), protoplast techniques are limited to a few genotypes; thus, the use of regular regeneration procedures of multicellular explants causes us to face complexities associated to CRISPR/Cas9 gene editing efficiency and final identification of individuals. Geminivirus-based replicons contained in T-DNAs could provide an improvement to these procedures considering their cargo capability. We built a Bean yellow dwarf virus-derived replicon vector, pGEF-U, that expresses all the editing reagents under a multi-guide RNA condition, and the Green Fluorescent Protein (GFP) marker gene. Agrobacterium-mediated gene transfer experiments were carried out on 'Yagana-INIA', a relevant local variety with no previous regeneration protocol. Assays showed that pGEF-U had GFP transient expression for up to 10 days post-infiltration when leaf explants were used. A dedicated potato genome analysis tool allowed for the design of guide RNA pairs to induce double cuts of genes associated to enzymatic browning (StPPO1 and 2) and to cold-induced sweetening (StvacINV1 and StBAM1). Monitoring GFP at 7 days post-infiltration, explants led to vector validation as well as to selection for regeneration (34.3% of starting explants). Plant sets were evaluated for the targeted deletion, showing individuals edited for StPPO1 and StBAM1 genes (1 and 4 lines, respectively), although with a transgenic condition. While no targeted deletion was seen in StvacINV1 and StPPO2 plant sets, stable GFP-expressing calli were chosen for analysis; we observed different repair alternatives, ranging from the expected loss of large gene fragments to those showing punctual insertions/deletions at both cut sites or incomplete repairs along the target region. Results validate pGEF-U for gene editing coupled to regular regeneration protocols, and both targeted deletion and single site editings encourage further characterization of the set of plants already generated.

VvDAM-SVPs genes are regulated by FLOWERING LOCUS T (VvFT) and not by ABA/low temperature-induced VvCBFs transcription factors in grapevine buds.

MAIN CONCLUSION: In deciduous fruit trees in which dormancy is induced by low temperatures, the expression of DORMACY-ASSOCIATED MADS-BOX genes (DAM) is regulated by CBF/DREB1 transcription factors. In Vitis vinifera, in which dormancy is induced by the photoperiod, VvDAM-SVPs gene expression is regulated by FLOWERING LOCUS T (VvFT). Using the sequences of the six peach (Prunus persica) DORMACY-ASSOCIATED MADS-box genes (DAM) as query, eight putative DAM genes belonging to the family of MADS-box transcription factors and related to the Arabidopsis floral regulators SHORT VEGETATIVE PHASE (SVP) and AGAMOUS LIKE 24 (AGL24) were identified in the V. vinifera genome. Among these, five belong to the subfamily SVP-like genes which have been associated with the regulation of flowering and dormancy in annual and perennial plants, respectively. It has been proposed that they play a direct role in the induction and maintenance of endodormancy (ED) through the regulation of the FLOWERING LOCUS T (FT) gene. In the present study, it is demonstrated that in V. vinifera: (1) VvDAM-SVPs genes are not regulated by ABA/low temperature-induced VvCBFs transcription factors as described for other species of deciduous fruit trees. (2) A contrasting expression pattern between VvDAM3-SVP and VvFT was found under different experimental conditions related to the entry and exit of grapevine buds from ED. (3) Overexpression of VvFT in somatic grapevine embryos (SGE) repressed the expression of VvDAM3-SVP and VvDAM4-SVP. Taken together, the results suggest that VvDAM3-SVP could be associated with ED in grapevine buds, and that its expression could be regulated by VvFT.

CRISPR/Cas9 Targeted Editing of Genes Associated With Fungal Susceptibility in Vitis vinifera L. cv. Thompson Seedless Using Geminivirus-Derived Replicons.

The woody nature of grapevine (Vitis vinifera L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system in vivo and evaluate their editing capability in individuals derived from Agrobacterium-mediated gene transfer experiments of 'Thompson Seedless' somatic embryos. Preliminary assays using a BeYDV-derived vector for green fluorescent protein reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including AUXIN INDUCED IN ROOT CULTURE 12 (VviAIR12), SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 4 (VviSWEET4), LESION INITIATION 2 (VviLIN2), and DIMERIZATION PARTNER-E2F-LIKE 1 (VviDEL1). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to Erysiphe necator and Botrytis cinerea. Assays have shown that a transgene-free VviDEL1 double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.

ABA Represses the Expression of Cell Cycle Genes and May Modulate the Development of Endodormancy in Grapevine Buds.

Recently, the plant hormone abscisic acid (ABA) has been implicated as a key player in the regulation of endodormancy (ED) in grapevine buds (Vitis vinifera L). In this study, we show that in the vine, the expression of genes related to the biosynthesis of ABA (VvNCED1; VvNCED2) and the content of ABA are significantly higher in the latent bud than at the shoot apex, while the expression of an ABA catabolic gene (VvA8H3) showed no significant difference between either organ. A negative correlation between the content of ABA and transcript levels of cell cycle genes (CCG) was found in both tissues. This result suggested that ABA may negatively regulate the expression of CCG in meristematic tissues of grapevines. To test this proposition, the effect of ABA on the expression of CCG was analyzed in two meristematic tissues of the vine: somatic embryos and shoot apexes. The results indicated that cell cycle progression is repressed by ABA in both organs, since it down-regulated the expression of genes encoding cyclin-dependent kinases (VvCDKB1, VvCDKB2) and genes encoding cyclins of type A (VvCYCA1, VvCYCA2, VvCYCA3), B (VvCYCB), and D (VvCYCD3.2a) and up-regulated the expression of VvICK5, a gene encoding an inhibitor of CDKs. During ED, the content of ABA increased, and the expression of CCG decreased. Moreover, the dormancy-breaking compound hydrogen cyanamide (HC) reduced the content of ABA and up-regulated the expression of CCG, this last effect was abolished when HC and ABA were co-applied. Taken together, these results suggest that ABA-mediated repression of CCG transcription may be part of the mechanism through which ABA modulates the development of ED in grapevine buds.

Relationship between endodormancy, FLOWERING LOCUS T and cell cycle genes in Vitis vinifera.

MAIN CONCLUSION: In grapevines, the increased expression of VvFT , genes involved in the photoperiodic control of seasonal growth ( VvAP1, VvAIL2 ) and cell cycle genes ( VvCDKA, VvCDKB2, VvCYCA1, VvCYCB, VvCYCD3.2 ) in the shoot apex relative to the latent bud, suggests a high mitotic activity of the apex which could prevent them to enter into endodormancy. Additionally, the up-regulation of these genes by the dormancy-breaking compound hydrogen cyanamide (H 2 CN 2 ) strongly suggests that VvFT plays a key role in regulating transcriptionally cell cycle genes. At the end of the growing season, short-day (SD) photoperiod induces the transition of latent grapevine buds (Vitis vinifera L) from paradormancy (PD) to endodormancy (ED), which allows them to survive the cold temperatures of winter. Meanwhile, the shoot apex gradually decreases its growth without entering into ED, and as a result of the fall of temperatures at the beginning of autumn, dies. To understand developmental differences and contrasting responses to environmental cues between both organs, the expression of cell cycle genes, and of genes involved in photoperiodic control of seasonal growth in trees, such as FLOWERING LOCUS T (FT), APETALA1 (AP1) and AINTEGUMENTA-like (AIL) was analyzed at the shoot apex and latent buds of vines during the transition from PD to ED. After shift to SD photoperiod, increased expression of cell cycle genes in the shoot apex suggests a high mitotic activity in this organ which could prevent them from entering into ED. Additionally, the increased expression of VvFT, VvAP1and VvAIL2 in the shoot apex, and the up-regulation of VvFT, VvAP1and cell cycle genes VvCDKA, VvCDKB2, VvCYCA.1, by the dormancy-breaking compound hydrogen cyanamide (H2CN2), strongly suggests that VvFT plays a key role in regulating transcriptionally cell cycle genes, giving thus, more support to the model for photoperiodic control of seasonal growth in trees. Furthermore, downregulation of VvFT by the SD photoperiod detected in leaves and buds of grapevines highlights the importance of VvFT in the induction of growth cessation and in ED development, probably by regulating the expression of cell cycle genes.

Evaluación in vitro de filtración hacia conducto radicular de dos sistemas de reconstrucción post endodóncica / In vitro assessment of root canal leakage of two post-endodontic reconstruction systems

El objetivo de este estudio fue el de evaluar el grado de microfiltración en dos sistemas de reconstrucción postendodóncica, valorando la penetración de un colorante a lo largo de las restauraciones realizadas con endopostes y cementos duales de resina. En este estudio se emplearon 30 dientes premolares unirradiculares extraídos a los que se les realizó tratamiento de conductos y almacenándolos en agua bidestilada a 37 ºC. Se distribuyeron los dientes en tres grupos de forma aleatoria, con diez dientes por cada grupo. La cementación de los postes se realizó en dos de los grupos, usando los sistemas RelyX Fiber post (3M ESPE dental products) y Parapost (Coltene-Whaledent). Las muestras fueron sometidas al proceso de termociclado y posterior inmersión en azul de metileno por siete días a 36 ºC. Posteriormente se diafanizaron las muestras y fueron analizadas bajo microscopio, obteniendo los valores de penetración en milímetros. Los resultados mostraron que los dientes tratados con el sistema Parapost presentaron menor microfiltración. El grupo control mostró el mayor índice de microfiltración. El análisis estadístico con la prueba de Donett mostró diferencia estadísticamente significativa entre los sistemas Parapost y RelyX. Conclusión: Se demostró que el sistema de reconstrucción Parapost (Coltene-Whaledent), el cual tiene una técnica de grabado ácido, reportó menores valores de microfiltración, siendo los resultados estadísticamente significativos con respecto a los obtenidos con el sistema de reconstrucción RelyX (3M ESPE dental products).

The aim of the present study was to assess the degree of microleakage observed when using two different post-endodontic reconstruction systems; this was achieved assessing penetration of a dye along restorations performed with endodontic posts and dual-cure resin cements. For the present study, 30 extracted, single rooted premolars were used. Root canal treatment was performed on the teeth which were later stored in bi-distilled water at 37 ºC. Teeth were randomly allotted to three groups, all groups were comprised of 10 teeth. In two groups, post cementation was performed using the systems RelyX Fiber Post (3M ESPE dental products) and Parapost (Coltene-Whaledent). Samples were subjected to thermo-cycling and later immersed in methylene blue for seven days at 36 ºC. After this, samples were subjected to a diaphanization process and were then analyzed under a microscope; penetration in millimeters was thus obtained. Results revealed that teeth treated with Parapost system exhibited lesser microleakage. Control group experienced the greater amounts of microleakage. Statistical analysis conducted with Donett test showed statistically significant difference between Parapost and RelyX systems. Conclusion: Parapost reconstruction system (Coltene-Whaledent) with acid-etch technique exhibited lower microleakage values. Results were statistically significant when compared to those obtained with the RelyX reconstruction system (3M ESPE dental products).

Hypoxia and hydrogen cyanamide induce bud-break and up-regulate hypoxic responsive genes (HRG) and VvFT in grapevine-buds.

It has been reported that dormancy-breaking compound hydrogen cyanamide (HC) stimulates the fermentative pathway and inhibits respiration in grapevine-buds, suggesting in this way, that a respiratory stress must be involved in the release of buds from dormancy. Here, we tested low-oxygen effect (hypoxia) on the bud-break response of endodormant grapevine buds, and HC and hypoxia effects on the expression of hypoxic responsive genes (HRG) PYRUVATE DECARBOXYLASE (VvPDC), ALCOHOL DEHYDROGENASE (VvADH2), SUCROSE SYNTHASE (VvSUSY), non-symbiotic HEMOGLOBIN (VvnsHb), and on FLOWERING LOCUS T (VvFT), a transcription factor related to dormancy release in Vitis. Hypoxia as HC, induce transiently the expression of HRG and VvFT and hasten the sprouting of endodormant grapevine-buds. During the first 24 h after treatment, HRG and VvFT were strongly induced by hypoxia, subsequently, their expressions fell, and 14 days post-treatment increased again above control levels. These results indicate that in the short-term, a respiratory stress, caused either by oxygen deprivation or by inhibitors of respiration, induces transiently the expression of HRG and VvFT, and in the long-term, along with the advancement of bud-break, the expression of these genes move forward in treated buds, suggesting that these second induction that occurs just before bud-break is developmentally regulated.

Hypoxia induces H2O2 production and activates antioxidant defence system in grapevine buds through mediation of H2O2 and ethylene.

Paradoxically, in eukaryotic cells, hydrogen peroxide (H(2)O(2)) accumulates in response to oxygen deprivation (hypoxia). The source of H(2)O(2) under hypoxia varies according to the species, organs, and tissue. In non-photosynthetic tissues, H(2)O(2) is mainly produced by activation of NAD(P)H-oxidases or by disruption of the mitochondrial electron transport chain (m-ETC). This study showed that hypoxia, and inhibitors of respiration like potassium cyanide (KCN) and sodium nitroprusside (SNP), trigger the production of H(2)O(2) in grapevine buds. However, diphenyleneiodonium, an inhibitor of NAD(P)H-oxidase, did not reduce the H(2)O(2) levels induced by KCN, suggesting that, under respiratory stress, H(2)O(2) is mainly produced by disruption of the m-ETC. On the other hand, γ-aminobutyric acid (GABA), a metabolite that in plants alleviates oxidative stress by activating antioxidant enzymes, reduced significantly the levels of H(2)O(2) induced by KCN and, surprisingly, repressed the expression of genes encoding antioxidant enzymes such as ASCORBATE PEROXIDASE (VvAPX), GLUTATHIONE PEROXIDASE (VvGLPX), SUPEROXIDE DISMUTASE (VvSOD), and one of the CATALASE isoforms (VvCAT1), while VvCAT2 was upregulated. In contrast to GABA, hypoxia, H(2)O(2), and ethylene increased dramatically the expression of genes encoding antioxidant enzymes and enzymes of the alternative respiratory pathway such as ALTERNATIVE NADH-DEHYDROGENASES (VvaNDs) and ALTERNATIVE OXIDASES (VvAOXs). Hence, it is concluded that H(2)O(2) production is stimulated by respiratory stress in grapevine buds, that H(2)O(2) and ethylene act as signalling molecules and activate genes related to the antioxidant defence system, and finally that GABA reduces H(2)O(2) levels by up-regulating the expression of VvCAT2.

Cirugia de la fractura de cadera en pacientes centenarios / Surgery of the hip fracture in centenarians patients

La incidencia de fractura de cadera ha aumentado, por envejecimiento de la población e incremento de su prevalencia en áreas urbanas. Cuando se presenta en pacientes centenarios, resulta un desafío quirúrgico del cual poco se ha reportado. El objetivo del presente trabajo es estudiar una pequeña casuística de ellos. Pacientes y método: se definió como paciente centenario, quien al presentar fractura de cadera tuviera 99 años cumplidos. Ellos fueron operados si su daño orgánico cerebral era leve, si no estaban postrados y no portaban patologías limitantes de su rehabilitación o sobrevida a 1año. Se revisó retrospectivamente la casuística del Servicio de Traumatología, entre 01/04/1999 y 3 1/03/2006. Se comparó sobrevida con datos estadísticos de población centenaria general en periodo estudiado. Resultados: se efectuaron 8 operaciones a seis pacientes, todas mujeres, edad promedio 100.2 años (rango 99-102). La comorbilidad más frecuente fue hipertensión arterial (3 de 6 casos) e infección urinaria ( 3/6). No hubo diabetes mellitus. En todos se usó anestesia raquídea, la cirugía consistió en reducción más inserción de tornillo dinámico de cadera en 5 casos, placa de compresióndinámica en 2 y prótesis parcial en una paciente. El promedio de hospitalización postoperatoria total fue 13,1 días (rango 5- 25), 2 presentaron complicaciones, no hubo mortalidad hospitalaria. La sobrevida fue similar a población general de igual edad sin fractura. Los resultados obtenidos permiten concluir que es posible abordar en nuestro medio, el desafío ético que significa tratar quirúrgicamente fracturas de caderas en pacientes centenarios.

The incidence of hip fracture has increased, due to population aging and augmentation of prevalence in urban areas. Centenarian patients suffering from hip fracture are a surgical challenge, with limited reporting in medical literature. The purpose of this paper is to communicate the results of small groupof them. Patients and method: all patients older than 99 year old were considered as centenarians.They were operated only if they did not suffer from dementia, prostration or limiting severe diseases that could diminish one year survival or full rehabilitation. Their record and chinical folder were studied retrospectively from 01/04/1999 until 31/03/2006 and survival compared with general centenarian population during the same period. Results: six patients underwent 8 operations, all were female, mean age 100,2 years (range 99-102). Three of 6 suffered from hipertensión, 3/6 from urinary infection. No diabetes was found. Surgery was done under regional anaesthesia, in 5 operations a dynamic hip screw was implanted, dynamic compression plate in 2 and parcial prosthesis was used in other. The average total postoperative time in the hospital was 13,1 days (range 5-25), 2 patients suffered from postoperative complications. There were no intrahospital mortality and survival was similar to the general centenarian population with no hip fractures. In view of this results, it is nowadays reasonable to face the ethical challenge to operate hip fractures in centenarian patients.

Síndrome del niño maltratado / Battered child syndrome

Se abordó el estudio del síndrome del niño maltratado (SNM) y de su fase superior: el síndrome del niño golpeado (SNG), para constituir ésta una primera etapa de análisis, con vista a la creación de un programa de prevención. Se creó una encuesta cerrada que recopiló los datos más importantes del desarrollo del niño y de su medio social, así como el cuadro clínico del síndrome. Se pudo detectar una alta incidencia del alcoholismo y las sociopatías en las familias de los pacientes, ausencia de relaciones armónicas, con predominio de las hostiles, bajo nivel cultural, desvinculación laboral, malas condiciones económicas, hacinamiento y promiscuidad, así como el no deseo del embarazo por las madres. En las sesiones de psicoterapia se constató que la actitud familiar negativa hacia el niño tiene carácter inconsciente. Se recomienda la generalización del conocimiento del SNM y del SNG entre los profesionales de la salud, para ampliar el diagnóstico y su tratamiento multidisciplinario al medio familiar

Síndrome del niño maltratado / Battered child syndrome

Se abordó el estudio del síndrome del niño maltratado (SNM) y de su fase superior: el síndrome del niño golpeado (SNG), para constituir ésta una primera etapa de análisis, con vista a la creación de un programa de prevención. Se creó una encuesta cerrada que recopiló los datos más importantes del desarrollo del niño y de su medio social, así como el cuadro clínico del síndrome. Se pudo detectar una alta incidencia del alcoholismo y las sociopatías en las familias de los pacientes, ausencia de relaciones armónicas, con predominio de las hostiles, bajo nivel cultural, desvinculación laboral, malas condiciones económicas, hacinamiento y promiscuidad, así como el no deseo del embarazo por las madres. En las sesiones de psicoterapia se constató que la actitud familiar negativa hacia el niño tiene carácter inconsciente. Se recomienda la generalización del conocimiento del SNM y del SNG entre los profesionales de la salud, para ampliar el diagnóstico y su tratamiento multidisciplinario al medio familiar (AU)