Different Concentrations Of Hormones Research Articles

Pioneering anther culture-based embryogenesis in Solanum aethiopicum L.

This study aimed to establish a compelling approach for inducing embryogenesis through in vitro anther culture in Scarlet eggplant (Solanum aethiopicum L.), the most consumed and popular eggplant among indigenous vegetables on the African continent. While in vitro androgenesis has been favorably employed in brinjal (Solanum melongena L.) breeding, there has been no attempt to induce embryogenesis in a large germplasm of its relative, Solanum aethiopicum. In two distinct experiments, the largest germplasm collection of Solanum aethiopicum gr. Gilo was assessed for embryogenesis induction using C medium supplemented with different concentrations of hormones. In the first experiment, callus induction was successful with an overall rate of 36.6 calli/100 anthers, but embryo formation was unsuccessful. Statistical analysis revealed a dependency of the rate of callus induction on accessions. In the second experiment, only four selected accessions of Solanum aethiopicum gr. Gilo were used and compared to two Turkish eggplant genotypes of Solanum melongena in two distinct treatments. The results showed that in the first treatment (I), only the accession GKE12 had a satisfactory outcome with a rate of embryo formation of 0.82/100 anthers and 0.41/100 anthers corresponding to the rate of developed embryos. In the second treatment (II), only controls, which were Adana and Kemer cultivars of Solanum melongena formed embryos with a rate of 7.26/100 anthers and 1.15/100 anthers, respectively. The obtained embryo/seedling of Solanum aethiopicum gr. Gilo was found to be diploid. Overall, this study demonstrated that with the right combinations of hormones, it is possible to induce embryogenesis and produce a diploid of Solanum aethiopicum, the world’s second most popular cultivated eggplant after brinjal. These findings could potentially contribute to the breeding of eggplants for enhanced genetic variation and resistance.

IN VITRO POTATO VARIABILITY AND ITS EFFECT ON TUBER NUTRITIONAL PROPERTIES

A comparative analysis of nine somaclonal Dutch selection “Alladin” potato variants test results was carried out using a modified Murashige-Skoog medium with different concentrations of hormones, vitamins and growth stimulants. As a result of the work carried out, 100% callus formation providing from leaf explants and more than 90% shoot formation without the use of additional steps associated with their rooting optimal protocols for in vitro cultivation of potatoes were defined. According to morphological characteristics 9 initial lines were selected among the Dutch selec-tion of “Alladin” potato variety regenerated plants using these protocols. The tubers of the second reproduction obtained from these lines were used for the analysis of bi-ochemical parameters (starch, soluble sugars, proteins). As a result, lines with the maximum starch content were identified, 2 lines out of 9 exceeded the control sample by 3.3% and 10.7%. The protein content in the tubers of 7 somaclonal variants from 1% to 10.6% exceeded this indicator in the control. In comparison to the control, the sugar content was significantly lower (from 37.9 to 54%) in 3 studied samples. The results obtained are of great practical interest for further breeding studies to consoli-date them and create new dietary and table varieties of potatoes. Based on the ob-tained somaclonal variants, new promising potato varieties adapted to the soil and climatic conditions of Northern Kazakhstan can be obtained.

Effects on Serum Hormone Concentrations after a Dietary Phytoestrogen Intervention in Patients with Prostate Cancer: A Randomized Controlled Trial.

Phytoestrogens have been suggested to have an anti-proliferative role in prostate cancer, potentially by acting through estrogen receptor beta (ERβ) and modulating several hormones. We primarily aimed to investigate the effect of a phytoestrogen intervention on hormone concentrations in blood depending on the ERβ genotype. Patients with low and intermediate-risk prostate cancer, scheduled for radical prostatectomy, were randomized to an intervention group provided with soybeans and flaxseeds (∼200 mg phytoestrogens/d) added to their diet until their surgery, or a control group that was not provided with any food items. Both groups received official dietary recommendations. Blood samples were collected at baseline and endpoint and blood concentrations of different hormones and phytoestrogens were analyzed. The phytoestrogen-rich diet did not affect serum concentrations of testosterone, insulin-like growth factor 1, or sex hormone-binding globulin (SHBG). However, we found a trend of decreased risk of increased serum concentration of estradiol in the intervention group compared to the control group but only in a specific genotype of ERβ (p = 0.058). In conclusion, a high daily intake of phytoestrogen-rich foods has no major effect on hormone concentrations but may lower the concentration of estradiol in patients with prostate cancer with a specific genetic upset of ERβ.

Preliminary results of in-vitro laboratory propagation of barberry (Berberis L.)

The genus Berberis vulgaris (Berberis L.) is currently found in tropical, subtropical, temperate, and cold climate zones. There is little doubt that Southeast Asia is where the majority of Berberis vulgaris' interspecies biodiversity is found. Additionally, species are widely distributed, mostly in hilly areas, and, in terms of moisture requirements, they are mesophytic and xeromesophytic plants. In this article, experiments on in-vitro propagation of Berberis vulgaris (Berberis L.) seedlings were carried out in laboratory conditions. During the experiments, the time of sterilization of the explants obtained from the Berberis vulgaris plant was determined. Different concentrations of hormones and auxins were used in several nutrient mediums for growing in artificial nutrient medium. The results depicted that the highest rate was observed in the variant, where 2ml.BAP+0.5NAA+30g sucrose+7g accounted for 87% of plant rooting rate. It was observed that the rooting rate was slightly higher in the control variant of WPM-Woody plant medium-nutrient medium than the control variant of the MS nutrient medium, which was 17%.

Potential benefits of amino acid supplementation for cervid performance and nutritional ecology, with special focus on lysine and methionine: A review.

Deer farming is a thriving industry for venison, velvet antlers, trophy hunting, and other by-products. Feeding and nutrition are important factors for improving production performance, especially dietary protein and amino acids (AAs), as they are the main components of all tissues. Only a few studies on AA supplementation (Lys, Met, Arg) have been performed on cervids, which show positive effects on weight gain, ADG, feed-:gain ratio, plasma AAs, carcass weight, dressing percentage, yield of high-quality muscles, storage of internal fat during winter, DM and CP digestibility, plasma protein- and fat-related metabolite concentrations, antler burr perimeter, weight, length and mineralisation, velvet antler yield, rumen volatile fatty acids, and microbiome composition. All these effects are relevant for supporting the production of cervids products, from venison to velvet or trophy antlers, as well as their general performance and well-being of captive-bred cervids. The current available information suggests that AA supplementation can be especially interesting for animals fed low protein rations, and growing animals, but should be avoided in high rations and during winter, since it may promote the accumulation of internal fat. Potential effects on milk production and the concentrations of different hormones involved in the regulation of the antler cycle should be further explored.

An Electric Signal Conduction Characterization Model (ESCCM) for Establishing an Effective Poplar Regenerative System

The poplar is a model system for research on wood plant biology. An establishment of an efficient poplar regeneration system (PRS) plays a key role in the molecular breeding of wood plants. At present, most established PRSs are based on orthogonal experiments of previous research data. However, such an experiment is complex, time-consuming, and inefficient for various poplar subspecies. Therefore, an efficient solution to the establishment of PRSs is urgent. In this study, the triploid white poplar (Populus tomentosa ‘YiXianCiZhu B385′) was used as an experimental material to establish a leaf-based regeneration system. Firstly, different concentrations of hormones were added into the medium for the differentiation, stretching, and rooting of leaves, and the electrical conductivity of the medium was measured by a conductivity meter. Secondly, the optimal hormone concentrations for differentiation, stretching, and rooting were obtained by wavelet analysis. Finally, the Electrical Signal Conduction Characterization Model (ESCCM) of different hormone concentrations in the differentiation, stretching, and rooting of poplars was established. The result showed that the ESCCM improves the efficiency of PRSs, and this provides new insight and theory in molecular breeding. The ESCCM also provides the possibility of an automated establishment of a PRS.

Numerical characterization of 1D-Photonic crystal waveguide for female reproductive hormones sensing applications

The current work investigates the detection of a wide range of estradiol and progesterone hormone concentrations by the use of a defective 1D photonic crystal. The suggested structure consists of repeating layers of PbS and SiO2, with a central cavity layer. The transfer matrix technique is manipulated to examine the transmission spectrum of the proposed structure. The mainstay of the present work is to analyse the shift in the wavelength of the defect mode concerning the changes in different concentrations of hormones that are infiltrated into the cavity layer. In the course of a simulation, various geometrical parameters such as the thickness of the cavity layer and incident angle are properly optimized to envisage high performance. The colour map plot is investigated to convey a clear view of the shift of defect mode position concerning the change in incident angle. Moreover, all the sensing parameters like sensitivity, figure of merit, quality factor, signal-to-noise ratio, sensor resolution, and limit of detection are evaluated for each hormone at different incident angles. The main application behind this study is that it is important to sense the levels of estradiol and progesterone hormones for balanced needs in a women's reproductive system as well as to monitor the overall health systems. This proposed structure is suitable for bio-sensing applications due to their cost-effective fabrication and high performance.

Basal blood concentrations of some orexigenic and anorexigenic hormones in obese and nonobese individuals according to blood groups.

Obesity is a serious public health problem associated with excessive food intake. Regulation of food intake in highly organized organisms is under the control of a large number of orexigenic and anorexigenic molecules. Therefore, the main purpose of this study has been to determine the relationship between obesity and some of the circulating orexigenic and anorexigenic peptides that have a role in appetite control and to determine whether the concentrations of these molecules differ according to blood groups. The study included 400 individuals of whom 100 were obese women, 100 obese men, 100 healthy men and 100 healthy women. Obese women and men were divided into 4 groups, according to their blood groups. In the control group, healthy women and healthy men were similarly divided into 4 blood groups. Each blood group within the groups, therefore, had 25 participants. When leptin, nesfatin-1, obestatin and neuropeptide-Y, ghrelin and galanin levels of the control group and obese participants were compared, regardless of blood groups, leptin, nesfatin-1, obestatin and neuropeptide-Y were significantly higher, whereas only the ghrelin levels were significantly lower in obese patients. When the amounts of these hormones were measured according to gender, the situation was similar. When leptin, nesfatin-1, obestatin and neuropeptide-Y values of the control and obese participants' blood groups were compared with each other; these hormones were high in all blood groups; however, leptin levels in A blood group, nesfatin-1 levels in AB and O blood group, obestatin levels in AB blood group, neuropeptide-Y levels in A, B, AB blood groups were significantly higher. When the ghrelin levels of the blood groups in the control group and obese participants were compared, it was only significantly lower in the AB blood group. The ghrelin levels in the other blood groups of the obese individuals were again low, but not significantly so. When the distribution of hormones according to gender was evaluated, a situation parallel to the above results was recorded. Leptin, nesfatin-1, obestatin and neuropeptide-Y and galanin levels of obese individuals were significantly higher than the control values, whereas the ghrelin values were significantly lower regardless of blood groups. Also, these hormones in blood partly varied with ABO blood groups. These different concentrations of hormones in ABO blood groups might be related with stimulation or suppression of appetite in human. However, further studies in other ethnic groups are needed to confirm these results.

Effect of cytokinins on growth and phenylpropanoid accumulation in Tartary buckwheat sprouts (Fagopyrum esculentum Moench)

This study analyzed the effect of plant hormones, zeatin, 6-benzyl amino purine (BAP), kinetin, and thidiazuron (TDZ) on the growth of Tartary buckwheat sprouts and analyzed the fresh weight, shoot and root length, and production of phenolic compounds. All the hormone-treated plants at the lowest concentration (0.1 mg/L) showed the highest levels of growth parameters (fresh weight, shoot, and root length) when compared to the control. Among the various hormones treatment, the plant treated with 1 mg/L of BAP, kinetin, and zeatin showed the highest total phenolic level, whereas the TDZ showed the highest accumulation of total phenolic at the lowest concentration (0.1 mg/L). A total of 6 compounds were identified (4-hydroxybenzoic acid, caffeic acid, chlorogenic acid, p-coumaric acid, rutin, and trans-cinnamic acid) were quantified by high liquid performance chromatography (HPLC) after treatment of plant with different concentrations of hormones. Among these individual phenolic compounds, at the higher hormonal concentration (1 mg/L) the rutin showed the highest accumulation in BAP, zeatin, and kinetin treated sprout, whereas in the TDZ treated sprout the rutin content was highest at the lowest concentration (0.1 mg/L). From these results, it is suggested that BAP, zeatin, and kinetin at the lowest concentrations might positively enhance the growth of buckwheat sprouts, whereas at the highest hormonal treatment the accumulation of the phenolic compounds was higher. However, in TDZ treatment the growth and phenolic compound accumulation were highest at the lowest concentration. From these results, it is showed that suitable concentrations might enhance the growth and phenolic compound accumulation in Tatary buckwheat sprout.

An efficient in vitro regeneration system from different wild apple (Malus sieversii) explants

BackgroundWild apple, Malus sieversii, is an endangered species and a valuable genetic resource that requires a variety of conservation techniques. This study aimed to investigate the influence of different concentrations of hormones on wild apple regeneration from leaf and stem explants to establish an optimal regeneration system.ResultsLeaves and stems derived from seedlings were cultured on several media supplemented with various concentrations of thidiazuron (TDZ) or 6-benzylaminopurine (BA) in different combinations with 1-naphthaleneacetic acid (NAA). The results showed that the most efficient shoot formation media (35% and 90%) were MS medium containing 4.0 mg L−1 TDZ and 1.0 mg L−1 NAA for leaf explants and MS medium containing 1.0 mg L−1 BA without NAA for stem explant. MS medium supplemented with 0.4 mg L−1 BA and 0.1 mg L−1 NAA (for shoot multiplication) and 1/2 MS + 0.1 mg L−1 NAA + 1.5% sucrose (for rooting) were effective media. Shoot regeneration from leaf explants was the most effective when the explants were placed abaxial side down onto the medium and were subjected to a pre-treatment of 3 weeks in darkness.ConclusionsAn optimized regeneration system for M. sieversii that allowed regeneration within 2–3 months developed. The protocol developed herein can be used in large-scale clonal propagation for the conservation of wild apple, M. sieversii.

Effects of Phytohormone Compatibility on Callus Growth of Rosa rugosa tunb

To obtain superior quality callus and to regenerate root system of Rosa rugosa tunb and improve culture efficiency, this experiment used a multi-factor orthogonal test method to establish a sterile system with young leaves of R. rugosa tunb as experimental materialsand different concentrations of hormones in each stage. Explore the effects of plant callus growth. The results showed that the optimal disinfection method of explants was 70% alcohol 30s+0.1% HgCl2 8min. At this time, the browning rate was 46.70%, and the infection rate was 10.00%, which was 14.40% and 72.67% lower than that before disinfection. [Primary medium]: MS+1.5 mg/L 6-BA+3.0 mg/L NAA+3% sucrose+0.9% agar, the induction rate at this time is 75.00%, an increase of 62.50% compared with no added hormone. [Proliferation medium] MS+2.0mg/L 6-BA+0.2mg/L NAA+3% sucrose+0.9% agar, the callus diameter reached 2.49cm after proliferation, the growth was strong, and the callus was highly available. [Rooting medium] MS+0.4mg/L IBA+0.5g/L AC+3% sucrose+ 0.9% agar, the rooting rate is 75.00%, the root system is long and the growth is robust.

Callus Formation on Endosperm from Immature and Mature Fruits of Barringtonia Racemosa

Barringtonia racemosa is mangroves type of plant which had been extensively utilized in conventional practices for relieving ailments of pain and inflammation. Many studies have been done on ethnobotanical profiles, pharmacological activities and chemical compounds in Barringtonia racemosa. However, there is a limited study on callogenesis of this plant particularly from different maturity stage of fruits. The present study is to identify the callogenesis of Barringtonia racemosa from endosperm explants of immature and mature fruits in MS medium supplemented with different concentrations of hormones 2,4-Dichlorophenoxyacetic acid (2,4-D) (0, 0.5, 1.0, 1.5 and 2.0 mg/L) and Kinetin (KIN) (0, 0.5, 1.0, 1.5 and 2.0 mg/L). The optimum hormone combination was found in callus grown on endosperm of immature fruits in MS medium supplemented with 1.5 mg/L 2,4-D and 1.0 mg/L KIN. It was also found that the callus in this treatment grew profusely with highest fresh weight (0.513 ± 0.022 g), 100% callus induction and friable callus texture. The callus fresh weight on endosperm explants was higher in immature fruits compared to mature fruits for all the hormone combinations. Therefore, callogenesis were found more efficient from endosperm explant of immature fruits in Barringtonia racemosa species.

Deciphering the transcriptomic insight during organogenesis in Castor (Ricinus communis L.), Jatropha (Jatropha curcas L.) and Sunflower (Helianthus annuus L.).

Cultivation of the castor crop is hindered by various factors and one of the approaches for genetic improvement of the crop is through exploitation of biotechnological tools. Response of castor tissues to in vitro culture is poor which necessitated this study on understanding the molecular basis of organogenesis in cultured tissues of castor, through de novo transcriptome analysis and by comparing with jatropha and sunflower having good regeneration ability. Transcriptome profiling analysis was carried out with hypocotyl explants from castor, jatropha and cotyledons from sunflower cultured on MS media supplemented with different concentrations of hormones. Differentially expressed genes during dedifferentiation and organogenic differentiation stages of callus included components of auxin and cytokinin signaling, secondary metabolite synthesis, genes encoding transcription factors, receptor kinases and protein kinases. In castor, many genes involved in auxin biosynthesis and homeostasis like WAT1, vacuolar transporter genes, transcription factors like short root like protein were down-regulated while genes like DELLA were up-regulated accounting for regeneration recalcitrance. Validation of 62 DEGs through qRT-PCR showed a consensus of 77.4% of the genes expressed. Overall study provides set of genes involved in the process of organogenesis in three oilseed crops which forms a basis for understanding and improving the efficiency of plant regeneration and genetic transformation in castor.

Effect of harmones on callus induction in Maize (Zea mays L.)

Callus induction from explants is a critical process in regeneration, micropropagation and transformation of maize (Zea mays L.) plants. Formation of callus from plant tissues on culture is affected by several factors. This study revealed to establish the effect of genotype, source of explants and auxin concentration on callus induction from five genotypes UMI 757 (G1), UMI 615 (G2), UMI 112 (G3), UMI 285 (G4) and CO 1 (G5) and one hybrid CO H (M) 5 (G6). Callus induction of the six maize varieties was investigated using immature embryos (E1), leaf bits (E2), root tips (E3), hypocotyls (E4) and seeds (E5) as explants with different concentrations of hormones. In this study, immature embryo was taken from 10 to 12 days after pollination (DAP) to get maximum response. The highest percentage of callus induction was observed (99.10) in immature embryo culture and seed culture gave the highest percentage of rhizogenic callus formation when compare to immature embryo. Among the genotypes tested, CO H (M) 5 recorded the highest callus induction percentage on (2D2K2) medium composition.

Development of an efficient In vitro Regeneration protocol in tomato (Solanum lycopersicum L.)

A protocol was developed for in vitro regeneration through somatic embryogenesis and organogenesis in tomato genotypes Roma, Punjab Ratta and Castle Rock. Hypocotyl and leaf explants were cultured on MS medium having different concentrations of hormones for callus induction. Maximum callogenesis from both hypocotyl and leaf explants was obtained on MS medium supplemented with 2, 4-D (2 mg/l) and Kin (0.5 mg/l). It was 86.66% for leaves and 70% for hypocotyls. Calli were cultured on MS medium having different concentrations of BAP (2–2.5 mg/l) and NAA (0.25–0.5 mg/l) for regeneration. But calli did not show any response for further callus growth and plant regeneration. However, in direct plant regeneration through organogenesis, shoot tip and hypocotyl explants were cultured on MS medium supplemented with Zeatin (1 mg/l) + IAA (0.1 mg/l) and BAP (2 mg/l) + NAA (0.2 mg/l). The maximum direct plant regeneration was obtained from both shoot tip and hypocotyl explants of genotype Punjab Ratta on MS medium supplemented with zeatin (1 mg/l) + IAA (0.1 mg/l). It was 81.66% for shoot tips and 73.33% for hypocotyls. Rooting was observed at half MS and 0.1 mg/l IBA. Rooted plantlets were made to acclimatize, hardened and gradually transferred to the pots.

Micropropagation of Caralluma adscendens var. fimbriata-An Indigenous Medicinal Plant of India

Present study describes micro-propagation of traditionally important medicinal plant in northern interior part of Karnataka, Caralluma adscendens var. fimbriata, for its conservation. Results showed that the survival ability of regenerated plants was 84 per cent. Proliferation of C. adscendens var. fimbriata shoots was achieved on medium supplemented with various concentrations of different hormones. The effective combination of 6-Benzylaminopurine (BA) 2.0 mg/l along with 0.5 mg/l kinetin (Kn) and 0.2 mg/l Indole-3-acetic acid (IBA) produced highest number of shoot sprouting frequency (91%) with a mean of 2.60 ± 0.17 shoot number per explants and a mean shoot length of 3.88 ± 0.20 cm from the mature nodal explants. A good frequency of callus was induced in 1.5 to 2.0 mg/l of 1-Naphthaleneacetic acid (NAA) and in 2.0 mg/l of 2,4-dichlorophenoxy acetic acid (2, 4-D) were transferred to root induction media. The NAA showed positive response of rooting when compared with IAA and IBA in the present study. The best root and shoot sprouted plants were successfully acclimatized in the greenhouse and transferred to open field conditions. This micropropagation protocol can be successfully used for large-scale multiplication and conservation of germplasm of this threatened plant.

Effect of various concentrations of different growth regulating hormones on callus weight and the amount of thymol of Thymus daenensis.

Thymus daenensisis a perennial plant belonging to Lamiaceae family that is a species endemic to Iran. Due to the indiscriminate use of this plant which possesses medicinal values, T. daenensis tissue culture is suggested to increase the quality and quantity of its effective compounds. Callus cultures using leaf and stem explants were subjected to different hormonal treatments. Ten-millimeter explants of leaves and stems were cut up from sterile seedlings. MS medium was used with different concentrations of naphthalene acetic acid, benzyl amino purine, kinetin and 2,4-D (mg per liter) in which callus formation was observed. The highest level of thymol (28/1335 micrograms per gram) was recorded in calluses derived from leaf explants whereas the lowest level was observed in calluses obtained from stem explants. Also the highest levels of thymol, i.e. 65/1313 and 30/1322 micrograms, were observed in treatments of 0.4 NAA+3 BAP and 0.8 NAA+6 BAP mg per liter, respectively. Moreover, the interaction of effects of different treatments of calluses and different concentrations of hormones showed that the highest level of thymol (38/1345 micrograms per gram) was obtained from the treatment of NAA+1.5 BAP 0.2 mg per liter in the calluses derived from leaf explants. In addition, the maximum callus weight (22.1 grams) was related to the treatment of 0.8 NAA+6 BAP milligrams per liter. Results of the callus culture of T. daenensis to enhance the quality and quantity of its active ingredients showed that different explants and hormones with different concentrations and as well as their interactions had a significant effect on the level of thymol and callus weight (p≤0.01).

In vitro Meristem Culture for Rapid Regeneration of Papaya Plantlets in Liquid Media

Papaya (Carica papaya L.) is an important crop in Kenya, produced both for local consumption and export. Papaya propagation via seeds makes it difficult in differentiating gender at seedlings phase. However, in vitro system could offer a solution to this problem. This study was an attempt to determine rapid in vitro methods for regeneration of papaya plantlets using liquid and semi solid/liquid double layer media and different concentration of growth regulators. Shoot meristems were harvested from hermaphrodite 30 cm tall papaya seedlings raised in greenhouse and cultured in Murashige and Skoog basal media supplemented with 30 g sucrose, and different concentrations of hormones which were treatments in liquid medium. The treatments comprised five levels (0, 0.05, 0.1, 0.15, and 0.2) of mg.L 2 Chloro 4 Pyridy – N Phenyl-urea (CPPU) in liquid and semi-solid/liquid double layer combination for shoot induction. For root initiation, four levels of Indole-3butyric acid (IBA) (0, 2.0, 2.5 and 3.0 mg.L) were used. The experiments were laid out in Original Research Article Gatambia et al.; ARRB, 9(1): 1-7, 2016; Article no.ARRB.22056 2 a completely randomized design with 3 replicates. Number of shoots, shoot length and leaf number were recorded every 2 weeks for 8 weeks. The percentage number of shoots that produced roots was also recorded. There was significant differences between the liquid and semi-solid /liquid media and the level of hormones used on shoot induction. The highest number of shoots (10), shoot length and number of leaves was recorded in the semisolid/ liquid double layer combination with CPPU at 0.15 mg.L. The highest root initiation (73.33%) was at 3.0 mg.L of IBA. Results indicated that semi-solid/liquid double layer combination could be used for rapid regeneration of papaya plantlets of known gender.

293 EFFECT OF DIFFERENT CONCENTRATIONS OF LH, FSH, AND E2 ON THE MATURATIONAL RATE OF INDIGENOUS SOUTH AFRICAN CATTLE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE STAINING

In vitro maturation of indigenous African cattle oocytes is a major challenge even though different maturation protocols work successfully in other breeds. The objective of this study was to determine the maturation rate of indigenous South African cattle oocytes following in vitro maturation in media supplemented with different concentrations of hormones and selected using brilliant cresyl blue (BCB) staining. Indigenous cattle ovaries were collected from the slaughterhouse and then oocytes were retrieved by aspiration method. A total of 966 oocytes were exposed to 26 µM BCB stain and 700 oocytes were not exposed to the BCB stain. Thereafter, oocytes exposed to the BCB stain were grouped according to the colour of their cytoplasm BCB+ (oocytes with blue cytoplasm, low G6PDH) and BCB– (unstained oocytes, increased G6PDH). The BCB exposed (BCB+ and BCB–) and the oocytes not exposed to BCB were then randomly allocated into tissue culture medium (TCM199) + 10% (vol/vol) fetal bovine serum (FBS) supplemented with 3 different concentrations of hormones as treatments (T). The T1 group was matured in the presence of 0.5 µg mL–1 of FSH, 5 mg mL–1 of LH, and 2 µg mL–1 of E2; the T2 group was matured in the presence of 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2; and the T3 group was matured in the presence of 1.5 µg mL–1 of FSH, 7 mg mL–1 of LH, and 4.5 µg mL–1 of E2. For IVM, 20 to 25 COC were placed in 50-µL droplets of IVM medium containing the 3 different levels of hormones. Maturation rate of oocytes was determined by the extrusion of the first polar body after 24 h of incubation in maturation medium. Data was analysed by ANOVA using SAS with 4 replicates per treatment. Treatment 2 yielded higher maturation rate for both BCB+ (65.6%) and not exposed to BCB (60.3%) oocytes compared to T1 (22, 3.03, and 16% for BCB+, BCB–, and not exposed to BCB, respectively) and T3 (48, 2.2, and 48% for BCB+, BCB–, and not exposed to BCB respectively). However, BCB– oocytes had lower polar body extrusion for T1, T2, and T3 (3.03, 8.1, and 2.2%, respectively) compared to BCB+ oocytes (22, 65.6, and 48% for T1, T2, and T3, respectively). In conclusion, immature oocytes that were cultured into TCM199 supplemented with 10% FBS, 1 µg mL–1 of FSH, 6 mg mL–1 of LH, and 2.5 µg mL–1 of E2 showed maturation rate for BCB+ oocytes and those not exposed to BCB. Oocytes selection using BCB staining was a useful test to classify good quality cattle oocytes. Therefore, it is suggested that treatment 2 is a suitable in vitro-maturation medium to mature indigenous South African cattle oocytes.

Assessment of DNA methylation changes in tissue culture of Brassica napus.

Plant tissue culture, as a fundamental technique for genetic engineering, has great potential of epigenetic variation, of which DNA methylation is well known of importance to genome activity. We assessed DNA methylation level of explants during tissue culture of Brassica napus (cv. Yangyou 9), using high-performance liquid chromatography (HPLC) assisted quantification. By detecting methylation levels in hypocotyls cultured in mediums with different concentrations of hormones, we found dissected tissue:cultured with 0.1 mg/L 2,4-D and 1.0 mg/L 6-BA, presented the lowest methylation level and highest induction rate of callus (91.0%). Different time point of cultured explants also showed obvious methylation variations, explants cultured after 6 and 21 days exhibited methylation ratios of 4.33 and 8.07%, respectively. Whereas, the methylation ratio raised to 38.7% after 30 days cultivation, indicating that methylation level of hypocotyls ranged during tissue culture. Moreover, we observed that the methylation level in callus is the highest during regeneration of rape-seed, following the regenerated plantlets and hypocotyls. This paper indicated the function of hormones and differentiation of callus is relevant to the methylation levels during tissue culture.