Two Agrobacterium-mediated Transformation Protocols of White Clover (Trifolium Repens) Through the Callus System

White clover (Trifolium repens) is one of the most widely cultivated livestock forage legumes co-cultivated worldwide with pasture grass in a mixed-sward setting, however, its persistence and aesthetic quality are severely affected by abiotic stresses. In this study, regeneration of white clover plants was conducted through a callus system for 4-5 months with a regeneration frequency of 36-41%. Inoculating 4-day-old cotyledons into MS media fortified with 0.4 mg·L-1 6-BA and 2 mg·L-1 2,4-D significantly increased the callus formation rate. Roots and cotyledons were better induced, followed by hypocotyls, leaves, and petioles. The development of differentiated structures performed effectively on MS supplemented with 1 mg·L-1 6-BA and 0.1 mg·L-1 NAA. Further, we determined factors affecting the Agrobacterium tumefaciens-mediated transient transformation for root-derived callus and 4-day-old cotyledons. The parameters that facilitated transient transformation were: Agrobacterium suspension density of 0.5 (OD600), 20 mg·L-1 AS, and 4-days co-cultivation duration. Subsequently, we developed two transformation protocols: transformation after callus formation in root segments (Protocol A) and transformation before callus initiation in 4-day-old cotyledons (Protocol B). The transformation frequencies varied from 1.92% to 3.17% in Protocol A and from 2.76% to 3.47% in Protocol B. We offer the possibility to regenerate multiple transgenic white clover from a single genetic background. In addition to assistance in identification of functional genes associated with yield, resistance and aesthetic quality, our research will also contribute to successful genetic manipulation and genome editing in white clover.

The Effect of Kryptonite Bone Cement combined with a Standard Kirschner Wire on Tibial Bone Fracture in Rats

Objective This study aims to observe whether Kryptonite Bone Cement combined with a standard Kirschner wire, instead of autogenous bone grafts, increases bone healing and mechanical strength in rats with tibia fractures. Methods The study included sixteen rats, which were divided into two groups as a control group (n=8) and an experimental group (n=8). After segmental fractures were made in both groups, intramedullary fixation of tibia procedures were conducted with the use of a Kirschner wire. No additional procedures were performed inthe control group, but Kryptonite Bone Cement was applied in the experimental group. The rats were evaluated clinically, radiologically and histologically4 times; immediately after the operation, and atthe 1st, 3rd, and 6thweeks following surgery. Results One rat from the experimental and one from the control group had both wound dehiscence and wound site infection. No recovery was observed in any rats either in the control or experimental group immediately after surgery. During week 1, callus formation was identified in 5 rats in the experimental group (p=0.0072), and during week 3, fracture lines disappeared in 4 rats in the experimental group (p=0.064); the differences between control and experimental groups were statistically significant. During week 6, no statistical significance was observed in radiological assessments for the control group and experimental group (p=0.71). The rate of non-union was higher in the control group (37.50%) than in the experimental one, while the rate of complete fusion was higher in the experimental group (87.50%) than that in the control group. Conclusion Osteoconductivity and ergonomic qualities of Kryptonite Bone Cement prove helpful in bone repair. Future studies to be conducted in a prospective and randomized manner will be effective on demonstrating the effectiveness of Kryptonite Bone Cement.

The effectiveness of agrotextile cover with organic photoluminophore in rooting cuttings of Hungarian lilac (Syringa josikaea J. Jacq. ex Rchb.)

The effectiveness of the use of a light-transforming shelter of vegetation structures consisting of a polypropylene spunbond with an organic photoluminophore integrated into its structure during the rooting of physiologically active stem cuttings of Hungarian lilac was studied. The object of the study was reproductively mature Hungarian lilac plants located in the arboretum of the Nizhny Novgorod State Agricultural Academy with geographical coordinates 56°14'32.7” N 43°57'20.7”E. The unequal reaction of the tested samples of Hungarian lilac to the use of luminophore in the shelters of vegetation structures during the rooting of cuttings was established, which manifested itself in all characteristics of regeneration processes. High rates of callus formation were in the shelter variants with a higher concentration of luminophore: 73.16 ± 5.95% and 65.25± 4.80%. Lower than in other shelters, the result was recorded in the variant with the lowest luminophore density: 47.00 ± 3.62%.

Induksi kalus daun binahong merah (Basella rubra L.) dengan pe,berian 2,4-D dan kinetin

Tanaman binahong merah (Basella rubra L.) merupakan salah satu tanaman yang mengandung senyawa metabolit sekunder berkhasiat obat. Kultur kalus adalah salah satu solusi dalam menghasilkan senyawa metabolit sekunder dengan jumlah yang besar. Penelitian ini bertujuan untuk mengetahui pengaruh pemberian 2,4-D dan kinetin dalam menginduksi kalus daun binahong merah. Penelitian dilakukan di Laboratorium Bioteknologi Tanaman Fakultas Pertanian Universitas Riau pada bulan November 2019 sampai Maret 2020. Percobaan menggunakan rancangan acak kelompok yang terdiri dari dua faktor yaitu 2,4-D dengan empat taraf konsentrasi yaitu 0 ppm, 0,5 ppm, 1 ppm dan 2 ppm dan kinetin dengan empat taraf konsentrasi yaitu 0 ppm, 0,5 ppm, 1 ppm dan 2 ppm dengan tiga ulangan. Hasil penelitian menunjukkan bahwa kombinasi perlakuan tanpa 2,4-D dan 0,5 ppm kinetin memberikan respon terbaik pada saat muncul kalus 11,67 HST dan perlakuan 1 ppm 2,4-D dan 2 ppm kinetin memberikan respon terbaik pada persentase keberhasilan induksi kalus 62,50 %. Red binahong (Basella rubra L.) is a plant that contains medicinal secondary metabolites. Callus culture is one solution in producing secondary metabolites in large quantities. This research aimed to determine the effect of 2,4-D and kinetin in inducing callus on red binahong leaves. The research was conducted at the Laboratory of Plant Biotechnology, Faculty of Agriculture, the University of Riau from November 2019 to March 2020. The experiment used a randomized block design with two factors, namely four levels of 2,4-D 0, 0.5, 1, and 2 ppm and four levels of kinetin namely 0, 0.5, 1, and 2 ppm with three replications. The results showed that a combination of 0 ppm 2,4-D and 0.5 ppm kinetin and 1 ppm 2,4-D and 2 ppm had the fastest callus formation 11.67 DAP and a combination of 1 ppm 2,4-D and 2 ppm kinetin produced weigher callus 6.4 mg and had a percentage of callus formation 62.50%.

Callus induction of mountain papaya endosperm (Vasconcellea pubescens A. DC) with different combination of 2,4-Dichlorophenoxyacetic acid and Kinetin

Endosperm as a result of double fertilization in Angiospermae shows high level chromosomes and polyploidy. It is also considered as dead tissue that unable to be generated to form plantlet. The aim of this research is to determine the effect of kinetin and 2,4-Dichlorophenoxyacetic acid (2,4-D) in induction of callus formation of mountain papaya. This research used A factorial randomized block design with 18 groups, 1 fruit was used for 1 experimental group. Culture using Murashige and Skog (MS) media with combination of three level of kinetin (0, 2, 4 mgL-1) and six level of 2,4-D (0, 1, 2, 3, 4, 5 mgL-1). Maximum endosperm callus induction (0.88%) was achieved from endosperm explant cultured on MS medium fortified with 2.0 mgL-1 Kinetin and 4.0 mgL-1 2,4-D. The fastest day induction (24,66 day) was observed with 5.0 mg L-1 2,4-D. The maximum number of browning (0,10) was induced by 2.0 mgL-1 Kinetin and 5.0 mgL-1 2,4-D. Combination Kinetin and 2,4-D proved could induces callus formation from mountain papaya endosperm.

In vitro propagation from nodal segments of Lippia origanoides (chemotype A)

ABSTRACT: This research described an efficient micropropagation protocol for Lippia origanoides (Verbenaceae). Sterile seeds were used to obtain germinated seedlings in Murashige and Skoog medium (MS) supplemented with sucrose and agar. The nodal segments obtained from seedlings were grown on MS medium supplemented with different concentrations of gibberellic acid (GA), benzylaminopurine (BAP) and 1-naphthalenacetic acid (NAA) with BAP. The callus induction, shoots length, shoots number and root length, were analyzed. The treatments showed high percentage of callus formation at 0.5 to 1.5 mg L-1 of BAP alone or in combination with NAA (0.1 mg L-1). The highest value of shoot number per nodal segments was obtained at 1.5 mg L-1 of BAP (4.3 ± 0.8). The obtained plantlets were better rooted in vitro in the absence of plant growth regulators (PGRs) and they showed acclimatization rate of 90%. We reported a protocol for in vitro propagation and acclimatization of L. origanoides for A chemotypes from Colombia.

Kultur suspensi sel tanaman gajah beranak (Goniothalamus tapis Miq) terhadap kandungan zat goniotalamin

Zat goniotalamin pada tanaman gajah beranak (Goniothalamus tapis) merupakan obat alternatif penyembuhan kanker. Penelitian bertujuan untuk mendapatkan zat goniotalamin melalui kultur kalus dan kultur suspensi sel. Metode penelitian eksperimen Rancangan Acak Lengkap (RAL) dengan kombinasi 2,4-D (1-10 mgL-1) dan BAP (0,5-2 mgL-1) menggunakan eksplan batang muda, terdiri dari 17 perlakuan dan 3 kali ulangan. Analisis data menggunakan Analysis of Variances dan uji lanjut Duncan Multiple Range Test (DMRT) taraf 5%. Hasil menunjukkan bahwa kultur kalus G. tapis pada media 5,0 mg L-1 2,4-D + 1 mg L-1 BAP adalah yang terbaik dengan waktu muncul kalus 28,33 hari dan persentase pembentukan kalus 100%. Kalus untuk kultur suspensi sel bertekstur remah dan berwarna kuning kehijauan. Kultur suspensi sel menghasilkan pertumbuhan sel yang cepat, tidak lembek berair dan mudah dipisahkan. Hasil kualitatif Kromatografi Lapis Tipis kultur suspensi sel sangat jelas, bersih dan terdapat potensi kandungan zat goniotalamin pada perlakuan 2,4-D 5 mg L-1 + BAP 0,5 mg L-1, 2,4-D 5 mg L-1 + BAP 1 mg L-1, 2,4-D 5 mg L-1 + BAP 2 mg L-1, 2,4-D 10 mg L-1 + BAP 0,5 mg L-1 dan 2,4-D 10 mg L-1 + BAP 1 mg L-1. Hasil kuantitatif zat goniotalamin dengan Kromatografi Cair Prestasi Tinggi terdapat pada perlakuan 2,4-D 5,0 mgL-1 + BAP 1 mg L-1 yaitu 9,57 mg g-1.The goniothalamine compound on Goniothalamus tapis is an alternative cancer medicine. This study aimed to obtain gonotalamin through callus culture and suspension cell culture. The experiment research method was Completely Randomized Design (CRD) with a combination of 2.4-D (1-10 mg L-1) and BAP (0.5-2 mg L-1) using young stem explants consisting of 17 treatments with 3 replications. Data analysis used ANOVA and DMRT at 5%. The results showed that G. tapis callus culture on 5.0 mg L-1 2.4-D + 1 mg L-1 BAP was the best treatment medium with callus emergence time of 28.33 days and percentage of callus formation 100%. The callus used for suspension cell culture was friable and greenish-yellow in color. Suspension cell culture resulted in rapid cell growth, was not fleshy, and easily separated. The quality test by Thin Layer Chromatography (TLC) from suspension cell culture resulted very clear, clean, and potential content of goniothalamin found in treatments 2.4-D 5.0 mg L-1 + BAP 0.5 mg L-1, 2.4-D 5.0 mg L-1 + BAP 1 mg L-1, 2.4-D 5.0 mg L-1 + BAP 2 mg L-1, 2.4-D 10 mg L-1 + BAP 0.5 mg-1 and 2.4-D 10 mg-1 + BAP 1 mg-1. The quantitative results of the best goniotalamine compounds in cell suspension cultures using High-Performance Liquid Chromatography (HPLC) on medium 2,4-D 5.0 mgL-1 + BAP 1 mg L-1 ie 9.57 g-1.

Response of black mulberry onto white mulberry rootstock to stenting (cutting-grafting) techniques and IBA concentrations

Ornamental black mulberry (Morus nigra L.) is used in landscape. Ornamental black mulberry which has high shade, tolerates air pollution and wind. White mulberry (Morus alba L.) is proper for kind of soils conditions. Therefore, this research was conducted to investigate the effects of stenting methods and IBA levels on some characters of black mulberry scions onto white mulberry rootstock for the first time. For this purpose, an experiment was carried out as factorial in completely randomized design with 10 replications. Two factors were including two stenting method (splice and omega) and three levels of Indole-3-butyric acid (0, 500 and 1000 mg L-1). Three months after grafting, percentage of rootstock callus formation, percentage of rooting, percentage of leaf formation, number of leaf formation, the longest shoot and the longest root formation were measured. Results showed that the effect of stenting method was significant on all of evaluated parameters and splice method was superior to omega method. Also, the effects of different levels of IBA were significant on all of measured traits and they are increased by increasing the levels of IBA and the best was with 1000 mg L-1. The interaction effects of these factors are significant on all of measured characteristics except for percentage of rootstock callus formation and rooting percentage. In conclusion, the stenting via splice method had higher success in comparison with omega method and the morphological traits increased by increasing the application of higher levels of IBA. The application of stenting method by splice plus 1000 mg L-1 IBA which was carried out for the first time is recommended for reproduction of ornamental black mulberry onto white mulberry.

Analysis of osteoblast, osteoclast levels and radiographic patterns in the healing process of bone fractures (preliminary research)

Objectives: The healing process of a bone fracture goes through many phases. The hard callus phase was critical where the original structure was conducted. The hard callus growth depends on osteoblasts and osteoclasts active, and this condition can be analyzed on the radiograph. This study aimed to examine the analysis of bone fracture healing between osteoblasts and osteoclast numbers and radiographic patterns. Materials and Methods: The study used 12 male Wistar rats with an incomplete fracture in the right femur made by a dental tapered bur with 0.3 mm in length and 0.2 mm in depth. Digital radiographic examinations were carried out on days 0, 5, 10, 17, and 25 after fracturing in a lateral position. Furthermore, a radiographic analysis was performed using Image-J to obtain changes in the value of length and depth in the healing area. The research was conducted to find the radiopaque and radiolucent patterns and the number of osteoblasts and osteoclasts. Results: This study resulted in a change in the radiograph pattern. Callus formation resulted in fracture areas with a smaller distance from day 0 to day 25. The bone healing process begins with granulation tissue formation, followed by the gradual replacement of the connective tissue and bone. This process is comparable to the increase in osteoblasts up to day 25, which blocks bone resorption. Osteoclasts regulate bone resorption, and their number increases after 10 and 17 days to replace bone formation. Osteoclasts decline after 25 days because osteoblasts inhibit them, which control bone formation. Conclusion: The conclusions were obtained there are changes in the radiograph pattern. The radiopaque increased while the radiolucent decreased; the osteoclast pattern tended to be stable and lowered while the osteoblasts increased during the fracture healing process. The correlation of all the factors is very closely related.