Bocconia frutescens L. induces neurological defects in rat offspring.

Nearly 80% of the world's population trusts traditional medicine and plant-based drug compounds to improve health, and more than 50% of women who participated in a study have used herbal remedies during pregnancy. Bocconia frutescens L. is a plant native to tropical America, where infusion of its leaves has been widely used for the treatment of several gastrointestinal disorders. We have already shown that orogastric consumption of B. frutescens L. during the organogenesis period at concentrations equivalent to human consumption produces teratogenic effects in rats, but effects on progeny development have not yet been studied. In this study, we aimed to investigate the possible association between the consumption of B. frutescens L. at a dose equivalent to that consumed by humans and the neurological development of rat progeny. Pregnant Wistar rats were administered lyophilized B. frutescens L. extract at 300 mg/kg/day or vehicle via the orogastric route during the organogenesis period (gestation days 7-13). The physical development and sensory and motor maturation of their offspring during lactation were analyzed with a battery of reflex and physical tests. B. frutescens L. produced a significant delay in physical development and sensorimotor maturation, compared to the control group. Proton nuclear magnetic resonance spectroscopy analysis showed signals for both flavonoids and alkaloids in the B. frutescens L. extract. We conclude that the delay in physical and neurological development could be interpreted as alterations in the maturation of some neuronal circuitries induced by B. frutescens L.

Multiplication of Cupressus guadalupensis Using the RITA® Temporary Immersion System.

The Guadalupe cypress (Cupressus guadalupensis S. Watson) is an endangered species included in the list of the NOM-059-SEMARNAT-2010. The presence of wild goats in the habitat has been the greatest threat to the propagation and survival of this species. Therefore, there is a need to generate propagation protocols that facilitate the regeneration of the species. Plant tissue culture offers various possibilities that can facilitate the regeneration of species under some risk. Temporary immersion systems have proven to be an option with various advantages in plant tissue culture, such as increasing the number of seedlings generated and reducing production times, compared to semisolid media. The objective of this chapter is to describe a protocol to propagate Guadalupe cypress tissues in a RITA® temporary immersion system.

Temporary Immersion System for Biomass Production of Salvia spp.: A Mini-Review.

Salvia is a very valuable medicinal plant for the pharmaceutical industry. Tissue culture techniques can be used to increase the number of plants in a shorter time. Although protocols for in vitro propagation of more than 15 plant species have been developed, they are not yet efficient enough to increase mass propagation of plants. Therefore, the use of temporary immersion systems is necessary to increase the morphological quality of plants as well as their biomass in several Salvia species. In this chapter, progress in in vitro propagation in several Salvia species using liquid medium and automation is described.

Selenium Injection in Dam Rabbits During Gestation Has Important Effects on Progeny Productive Performance.

Micromineral elements have a fundamental participation in the processes of organogenesis and fetal development. The objective of this study was to assess the effect of selenium (Se) injection in pregnant female rabbits, during organogenesis or rapid fetal growth, on the productive performance of their progeny. An experiment was carried out with 30 New Zealand female rabbits, with an average age of 6 months. At the end of mating (day 0), using a randomized complete design, the female rabbits were distributed into three experimental groups, which were assigned to the following treatments: Control, female rabbits were injected intramuscularly (IM) with 0.5 ml of saline on days 13 and 23 of gestation; Early administration, female rabbits that were injected IM with Se (0.10 mg/kg BW) on day 13 of gestation (organogenesis) and 0.5 ml of saline on day 23 of gestation; and Late administration, female rabbits that were injected IM with 0.5 ml of saline on day 13 of gestation and Se (0.10 mg/kg BW) on day 23 of gestation (rapid fetal growth). No differences were found on kindling performance of dams and pre-weaning growth of rabbit offspring. However, an injection of Se to pregnant rabbits affected the growth and development of their progeny, with the treatment leading to changes in the yield of some carcass traits (forelimb weight and forelimb muscle weight) and weights of some organs (liver, lungs, and spleen). The Se treatment (both early and late) also resulted in lower concentrations of glucose, triglycerides, and cholesterol when compared to the control group. These effects were different when Se injection was performed during organogenesis or rapid fetal development. The results from this study suggest that there are beneficial effects of gestational Se injection of rabbit dams on important productive traits of their progeny.

Effect of the Aqueous Extract of Chrysobalanus icaco Leaves on Maternal Reproductive Outcomes and Fetal Development in Wistar Rats.

Toxicological studies on medicinal plants are essential to ensure their safety and effectiveness in treating various diseases. Despite the species Chrysobalanus icaco L. being popularly used in the treatment of several diseases due to the pharmacological properties of its bioactive compounds, there are few studies in the literature regarding its toxicity regarding reproduction. Therefore, the purpose of this study was to assess the potential embryotoxic and teratogenic effects of the aqueous extract of C. icaco leaves (AECi) on Wistar rats. Animals were given AECi at doses of 100, 200, and 400 mg/kg during the pre-implantation and organogenesis periods. Data were analyzed using ANOVA followed by Tukey's test and Kruskal-Wallis. Pregnant rats treated during the pre-implantation period showed no signs of reproductive toxicity. Rats that received AECi at 100, 200, and 400 mg/kg during organogenesis did not exhibit any signs of maternal systemic toxicity or significant differences in gestational and embryotoxic parameters. Some skeletal changes were observed in the treated groups. Therefore, it can be suggested that AECi at doses of 100, 200, and 400 mg/kg is safe for treated animals and does not induce reproductive toxicity under the experimental conditions applied, but it also caused low systemic toxicity.

Maternal diabetes increases FOXO1 activation during embryonic cardiac development.

Maternal diabetes is known to affect heart development, inducing the programming of cardiac alterations in the offspring's adult life. Previous studies in the heart of adult offspring have shown increased activation of FOXO1 (a transcription factor involved in a wide variety of cellular functions such as apoptosis, cellular proliferation, reactive oxygen species detoxification, and antioxidant and pro-inflammatory processes) and of target genes related to inflammatory and fibrotic processes. In this work, we aimed to evaluate the effects of maternal diabetes on FOXO1 activation as well as on the expression of target genes relevant to the formation of the cardiovascular system during organogenesis (day 12 of gestation). The embryonic heart from diabetic rats showed increased active FOXO1 levels, reduced protein levels of mTOR (a nutrient sensor regulating cell growth, proliferation and metabolism) and reduced mTORC2-SGK1 pathway, which phosphorylates FOXO1. These alterations were related to increases in the levels of 4-hydroxynonenal (an oxidative stress marker) and increased mRNA levels of inducible nitric oxide synthase, angiopoietin-2 and matrix metalloproteinase-2 (MMP2) (all FOXO1 target genes relevant for cardiac development). Results also showed increased extracellular and intracellular immunolocalization of MMP2 in the myocardium and its projection into the lumen of the cavity (trabeculations) together with decreased immunostaining of connexin 43, a protein relevant for cardiac function that is target of MMP2. In conclusion, increases in active FOXO1 induced by maternal diabetes initiate early during embryonic heart development and are related to increases in markers of oxidative stress and of proinflammatory cardiac development, as well to an altered expression of proteolytic enzymes that regulate connexin 43. These alterations may lead to an altered programming of cardiovascular development in the embryonic heart of diabetic rats.

Embryo-fetal safety evaluation of ondansetron in rats.

BACKGROUND: Ondansetron is a 5HT3 receptor antagonist, used to mitigate the effects of nausea and vomiting after chemotherapy or surgery. Since nausea and vomiting are common experiences during the first trimester of pregnancy, this antiemetic has been the main drug used during this period. METHODS: To evaluate the effects of ondansetron on the embryo-fetal development, which are still very contradictory, pregnant rats were exposed to therapeutic doses of ondansetron (1.7 or 2.5 mg/kg) daily, from gestational day (GD) 6 to 15. RESULTS: No clinical signs of toxicity were observed in dams during the treatment. Although the hemato-biochemical parameters were similar among the groups, histological changes, as well as a reduction in the weight of kidney were found in the treated dams. After fetal examination, no visceral and skeletal abnormalities were observed in treated fetuses. CONCLUSION: In conclusion, therapeutic doses of ondansetron have low teratogenic potential in rats. These data provide important information about the drug safety during pregnancy.

Characterization and Expression of Holothurian Wnt Signaling Genes during Adult Intestinal Organogenesis.

Wnt signaling has been shown to play multiple roles in regenerative processes, one of the most widely studied of which is the regeneration of the intestinal luminal epithelia. Most studies in this area have focused on self-renewal of the luminal stem cells; however, Wnt signaling may also have more dynamic functions, such as facilitating intestinal organogenesis. To explore this possibility, we employed the sea cucumber Holothuria glaberrima that can regenerate a full intestine over the course of 21 days after evisceration. We collected RNA-seq data from various intestinal tissues and regeneration stages and used these data to define the Wnt genes present in H. glaberrima and the differential gene expression (DGE) patterns during the regenerative process. Twelve Wnt genes were found, and their presence was confirmed in the draft genome of H. glaberrima. The expressions of additional Wnt-associated genes, such as Frizzled and Disheveled, as well as genes from the Wnt/ß-catenin and Wnt/Planar Cell Polarity (PCP) pathways, were also analyzed. DGE showed unique distributions of Wnt in early- and late-stage intestinal regenerates, consistent with the Wnt/ß-catenin pathway being upregulated during early-stages and the Wnt/PCP pathway being upregulated during late-stages. Our results demonstrate the diversity of Wnt signaling during intestinal regeneration, highlighting possible roles in adult organogenesis.

Pronephros and mesonephros characterization during the embryonic development of the giant South American river turtle, Podocnemis expansa (Podocnemididae: Testudines).

This study aimed to describe pronephros and mesonephros morphology during the embryonic development of Podocnemis expansa. Eggs were collected on an artificial beach at Balbina, Amazonas, Brazil, during the entire incubation period (mean of 59 days). The kidney-gonad complex was processed using light microscopy and the mesonephros using transmission electron microscopy. The pronephros was present for the first time on stage 4, composed of external glomeruli devoid of a capsule, protruding into the coelomic cavity, and internally composed of a capillary network. The pronephros degenerated after development stage 15. The first sign of the appearance of the mesonephros occurred around stage 8, indicated by the early formation of renal corpuscles. The mesonephros comprised an renal corpuscles, neck segment, proximal tubule, intermediate segment, distal tubule, collector tubule, and collector duct. Ultrastructural analysis of the mesonephros brush border was done in the proximal tubule, and the presence of cells with structural characters indicative of secretory activity was detected in the juxtatubular region. Renal corpuscles and proximal tubules were the main components that underwent morphological alterations during mesonephros degeneration. The pronephros is a transient kidney, and the mesonephros became the functional embryonic kidney in P. expansa. Mesonephros degeneration occurs in the cranial-caudal direction, and histologically, the degeneration is identified by changes in the morphology of the renal corpuscle and proximal tubule. However, the mesonephros is still present after hatching.

Effects of Maternal Protein Supplementation at Mid-Gestation of Cows on Intake, Digestibility, and Feeding Behavior of the Offspring.

This study aimed to assess the effects of maternal protein supplementation and offspring sex (OS) on the intake parameters of the offspring. Forty-three Tabapuã cows were randomly allocated in the following treatments: protein supplementation (PS) during days 100-200 of gestation (RES, 5.5% total crude protein (CP), n = 2, or CON, 10% total CP, n = 19) and OS (females, n = 20; males, n = 23). The offspring were evaluated during the cow-calf (0-210 days), backgrounding (255-320 days), growing 1 (321-381 days), and growing 2 (382-445 days) phases. The CON offspring tended to present higher dry matter intake (DMI) at weaning (p = 0.06). The CON males presented lower digestibility of major diet components in the growing 2 phase (p ≤ 0.02). The CON offspring spent 52% more time per day eating supplements at 100 days and 17% less time in idleness at 210 days. The CON males spent 15 min more per day ruminating than RES males in the feedlot phase (p = 0.01). We concluded that protein supplementation over gestation alters the offspring feed intake pattern as a whole, while protein restriction promotes compensatory responses on nutrient digestibility in males.

Advances in the Micropropagation and Genetic Transformation of Agave Species.

The Agave genus is composed of approximately 210 species distributed from south United States to Colombia and Venezuela. Numerous Agave species have been used for the preparation of alcoholic beverages and have attracted interest in the pharmaceutical and food industry. Despite their economic importance, there are few initiatives for the improvement and selection of characteristics of interest. This is mainly due to its morphology, long lifecycles, and monocarpic nature. Micropropagation is a feasible alternative to the improvement of Agave species. It has been used for multiple purposes, including massive propagation, induction of somaclonal variation to enhance agronomic characteristics of interest, maintenance of specific genotypes, and genetic transformation using molecular techniques. In this report, we summarize the most outstanding findings regarding the micropropagation of Agave species mediated by multiple regeneration responses. We also describe the media and growth regulators for each of the previously described methods. In addition, we discuss how micropropagation has allowed the development of transformation protocols. Exploitation of this technology may be a feasible strategy to introduce genes and improve certain traits. Genetic transformation also offers an opportunity for studying molecular mechanisms. This represents advantages for optimizing production in the field and for implementing breeding programs.

High-definition ultrasonography for evaluation of embryonic and fetal development in bitch / Ultrassonografia de alta-definição para avaliação do desenvolvimento embrionário e fetal em cadelas

In women, high-definition ultrasonography provides high-quality images of normal and diseased tissues throughout pregnancy, however there are no veterinary studies describing the use of this technology in pregnant dogs. The objective was to evaluate pregnant female dogs daily, using high-definition ultrasonography (HD) in brachycephalic, documenting daily findings of maternal-fetal structures. Twelve healthy brachycephalic females were evaluated daily by HD ultrasound until delivery. It was possible to identify and categorize all maternal, embryonic, and fetal structures and their development, including some fetal tissues not yet described in the literature. The HD exam is an excellent method to monitor the pregnancy of female dogs and identify structures early, providing safe and complete information regarding fetal development.

Em mulheres, a ultrassonografia de alta definição fornece imagens de alta qualidade de tecidos normais e alterados durante a gravidez, entretanto não existem estudos veterinários que descrevam o uso dessa tecnologia em cadelas prenhas. O objetivo deste estudo foi avaliar diariamente cadelas braquicefálicas gestantes, por meio de ultrassonografia de alta definição (HD), documentando achados diários das estruturas materno-fetais. Doze cadelas braquicefálicas saudáveis foram avaliadas diariamente por ultrassonografia HD, desde a inseminação até o parto. Foi possível identificar e categorizar todas as estruturas maternas, embrionárias e fetais e seu desenvolvimento, incluindo alguns tecidos fetais ainda não descritos na literatura. O exame de HD é um excelente método para monitorar a gestação de cadelas e identificar precocemente as estruturas, fornecendo informações seguras e completas sobre o desenvolvimento fetal.

Spontaneous Regeneration of Plantlets Derived from Hairy Root Cultures of Lopezia racemosa and the Cytotoxic Activity of Their Organic Extracts.

A histological analysis was performed with the aim of elucidating the spontaneous regeneration process of the hairy root lines LRT 2.3 and LRT 6.4, derived from Lopezia racemosa leaf explants and genetically transformed with the Agrobacterium rhizogenes strain ATCC15834/pTDT. The analysis showed both lines regenerate via indirect somatic embryogenesis; LRT 6.4 also regenerated by direct organogenesis. The morphogenic characteristics of the regenerated plantlets from both lines showed the typical characteristics, described previously, including a higher number of axillary shoot formation, short internodes, and plagiotropic roots compared with wild-type seedlings. The regeneration process occurred without the addition of plant growth regulators and was linked to the sucrose concentration in the culture medium. Reducing the sucrose concentration from 3% to 2%, 1%, and 0.5% increased the regeneration rate in LRT 6.4; the effect was less pronounced in LRT 2.3. The cytotoxic activity of different organic extracts obtained from roots and shoots were evaluated in the cancer cell lines HeLa (cervical carcinoma), HCT-15 (colon adenocarcinoma), and OVCAR (ovary carcinoma). The hexane and dichloromethane extracts from roots of both lines showed cytotoxic activity against the HeLa cell line. Only the dichloromethane extract from the roots of PLRT 2.3 showed cytotoxic activity against the OVCAR cell line. None of the methanol extracts showed cytotoxic activity, nor the shoot extracts from any solvent.

Female Prostate Development: Morphological Analysis of the Budding Dynamic.

The presence of the prostate in female mammals has long been known. However, pieces of information related to its development are still lacking. The aim of this study was to explore the budding dynamic during the initial prostate development in female gerbils. Pregnant females were timed, the fetuses were euthanized, and the urogenital sinus was dissected out between the embryonic days 20 and 24 (E20-E24 groups). Newborn pups (1-day-old; P1 group) underwent the same procedures. The female prostate development was based on epithelial buds which arose far from the paraurethral mesenchyme (PAM). The epithelial buds reached the PAM at prenatal day 24, crossing a small gap in the smooth muscle layer between the periurethral mesenchyme (PEM) and the PAM. Steroid nuclear receptors such as the androgen receptor and estrogen receptor alpha were localized in the PEM through the urethral wall, although some epithelial labeling was also present in the urogenital sinus epithelium (UGE). P63-positive cells were found only in the UGE, becoming restricted to the basal compartment after the 23rd prenatal day. The results showed that the gerbil female prostate exhibits a distinct budding pattern as compared to the male prostate development.

Endocytic trafficking induces lateral root founder cell specification in Arabidopsis thaliana in a process distinct from the auxin-induced pathway.

Plants can modify their body structure, such as their root architecture, post-embryonically. For example, Arabidopsis thaliana can develop lateral roots as part of an endogenous program or in response to biotic and abiotic stimuli. Root pericycle cells are specified to become lateral root founder cells, initiating lateral root organogenesis. We used the endocytic trafficking inducer Sortin2 to examine the role of endomembrane trafficking in lateral root founder cell specification. Our results indicate that Sortin2 stimulation turns on a de novo program of lateral root primordium formation that is distinct from the endogenous program driven by auxin. In this distinctive mechanism, extracellular calcium uptake and endocytic trafficking toward the vacuole are required for lateral root founder cell specification upstream of the auxin module led by AUX/IAA28. The auxin-dependent TIR1/AFB F-boxes and auxin polar transport are dispensable for the endocytic trafficking-dependent lateral root founder cell specification; however, a different set of F-box proteins and a functional SCF complex are required. The endocytic trafficking could constitute a convenient strategy for organogenesis in response to environmental conditions.

An Eye in the Replication Stress Response: Lessons From Tissue-Specific Studies in vivo.

Several inherited human syndromes that severely affect organogenesis and other developmental processes are caused by mutations in replication stress response (RSR) genes. Although the molecular machinery of RSR is conserved, disease-causing mutations in RSR-genes may have distinct tissue-specific outcomes, indicating that progenitor cells may differ in their responses to RSR inactivation. Therefore, understanding how different cell types respond to replication stress is crucial to uncover the mechanisms of RSR-related human syndromes. Here, we review the ocular manifestations in RSR-related human syndromes and summarize recent findings investigating the mechanisms of RSR during eye development in vivo. We highlight a remarkable heterogeneity of progenitor cells responses to RSR inactivation and discuss its implications for RSR-related human syndromes.

Organogenesis on apical buds in common fig (Ficus carica) var. Black Jack

BACKGROUND Plant tissue culture involves the use of explants obtained from plants to induce organogenesis with the help of plant growth regulators (PGRs). Micropropagation techniques provide a faster and economical solution to the limitations associated with traditional methods of plant cultivation. The present study focuses on the multiple shoot induction and proliferation of Ficus carica var. Black Jack. Factors that influence the growth of in vitro multiple shoots on the apical buds, which include growth media and PGRs, were investigated in this study. Different concentrations of cytokinins like 6-benzylaminopurine (BAP), Thidiazuron (TDZ), and Kinetin (Kin) were used on woody plant medium (WPM) for the optimization of media for multiple shoot induction and proliferation. RESULTS Apical buds of Ficus carica var. Black Jack growing in WPM supplemented with BAP produced the healthiest plantlets, with the highest number of multiple shoots. The most efficient medium composition which produced the highest number of multiple shoots (37.8) per growing explant was WPM supplemented with 20 mM BAP. Proliferated multiple shoots were efficiently rooted using WPM + 20 mM BAP + 8 mM indole-3-acetic acid (IAA). This optimized medium composition significantly enhanced the production of multiple, disease-free plantlets using single apical bud explants of Ficus carica var. Black Jack. CONCLUSIONS In the present study the observations indicate that WPM supplemented with 20 mM BAP is the best-suited medium for organogenesis and multiple shoot culture of Ficus carica var. Black Jack, and this technique can be potentially applied for commercialization of the plant

Differential regulation of transposable elements (TEs) during the murine submandibular gland development.

The submandibular gland (SG) is a relatively simple organ formed by three cell types: acinar, myoepithelial, and an intricate network of duct-forming epithelial cells, that together fulfills several physiological functions from assisting food digestion to acting as an immune barrier against pathogens. Successful SG organogenesis is the product of highly controlled and orchestrated genetic and transcriptional programs. Mounting evidence links Transposable Elements (TEs), originally thought to be selfish genetic elements, to different aspects of gene regulation in mammalian development and disease. To our knowledge, the role of TEs during murine SG organogenesis has not been studied. Using novel bioinformatic tools and publicly available RNA-Seq datasets, our results indicate that a significant number of genic and intergenic TEs are differentially expressed during the SG development. Furthermore, changes in expression of specific TEs correlated with that of genes involved in cellular division and differentiation, critical aspects for SG maturation. Altogether, we propose that TEs modulate gene networks that operate during SG development.

Transcription Landscape of the Early Developmental Biology in Pigs.

Since pre- and postnatal development are programmed during early prenatal life, studies addressing the complete transcriptional landscape during organogenesis are needed. Therefore, we aimed to disentangle differentially expressed (DE) genes between fetuses (at 35 days old) and embryos (at 25 days old) through RNA-sequencing analysis using the pig as model. In total, 1705 genes were DE, including the top DE IBSP, COL6A6, HBE1, HBZ, HBB, and NEUROD6 genes, which are associated with developmental transition from embryos to fetuses, such as ossification, skeletal muscle development, extracellular matrix organization, cardiovascular system, erythrocyte differentiation, and neuronal system. In pathway analysis, embryonic development highlighted those mainly related to morphogenic signaling and cell interactions, which are crucial for transcriptional control during the establishment of the main organs in early prenatal development, while pathways related to myogenesis, neuronal development, and cardiac and striated muscle contraction were enriched for fetal development, according to the greater complexity of organs and body structures at this developmental stage. Our findings provide an exploratory and informative transcriptional landscape of pig organogenesis, which might contribute to further studies addressing specific developmental events in pigs and in other mammals.

Micropropagación de Calibanus hookeri (lem.) trel (1911). Una especie amenazada / Micropropagation of Calibanus hookeri (lem.) trel (1911). A threatened species

RESUMEN La especie Calibanus hookerii perteneciente a la familia Asparagaceae, está registrada en la NOM-059-SEMARNAT-2010 catalogada como planta amenazada. Sus poblaciones naturales se han visto reducidas de manera importante debido a una explotación excesiva y destrucción de su hábitat, por lo que se requiere de métodos de propagación eficaz que aseguren su conservación. La propagación in vitro es una alternativa viable para especies vegetales amenazadas. En la presente investigación se reporta el protocolo para la micropropagación de Calibanus hookerii mediante la germinación de semilla sin testa inoculada en medio MS complementado con 2.5, 5.0 y 7.0 mg L-1 de 6 benciladenina (BA), cinetina (K), 2-isopentil-adenina (2iP) y tidiazuron (TDZ). Las variables a medir fueron porcentaje de germinación, número y longitud de brotes producidos por semilla. El tratamiento más eficiente fue de 5.0 mg L-1 de BA produciéndose un promedio de 26 brotes por semilla; el tratamiento menos eficaz fue con 2.5 mg L-1 K en el cual solamente se obtuvieron dos brotes por semilla. De las tres concentraciones de 2iP solamente en la concentración de 7 mg. L-1 mostró resultados produciendo 6 brotes por semilla. En lo que respecta a la longitud del brote ningún tratamiento superó al testigo (8.07cm). La eficiencia la germinación in vitro fue de 56-97%.

ABSTRACT The species Calibanus hookerii belonging to the family Aspagaceae is registered in the NOM-059-SEMARNAT-2010 cataloged in danger of extinction and therefore is necessary of propagation methods that assure its conservation and its propagation. In vitro propagation is a viable alternative for endangered plant species. The present investigation reports the protocol for the micropropagation of Calibanus hookerii. This was achieved by seed germination without test in MS medium supplemented with 2.5, 5.0 and 7.0 mg L-1 of 6-benzyladenine (BA), kinetin (K), 2-isopentyl-adenine (2iP) and tidiazuron (TDZ). The variables to be measured were percentage of germination, number and length of shoots produced by seed. The most efficient treatment was 5 mg L-1 of BA producing an average of 26 shoots per seed, the worst treatment was with 2.5 K only produced 2 shoots per seed, of the four cytokinins used 2ip treatment in only one study of the Performed showed results (7 mg L-1) producing 6 shoots per seed. Regarding the length of the shoot, no treatment exceeded the control (8.07cm). Finally, in vitro germination was high (56-97%) in all treatments.