Efficient Plant Regeneration via Indirect Organogenesis in Carnation (Dianthus Caryophyllus Semperflorens flore pleno) Cultivars

ABSTRACT Callus induction and plant regeneration are important steps of in vitro plant breeding of ornamental plants. In this study, the effects of different combinations of plant growth regulators (PGRs), promoters, and minerals on callus induction and plant regeneration in different carnation cultivars were studied in a completely randomized design with three replications. For callus induction, 16 different combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA), 1-naphthaleneacetic acid (NAA), and casein hydrolysate (CH) were studied using in vitro leaf explants. The Murashige and Skoog (MS) medium supplemented with 0.2 mg·dm-3 of 2,4-D and 200 mg·dm-3 of CH showed the highest frequency of callus induction. Among the cultivars, ‘Noblesse’ showed the highest rate of callus induction (91.67%). Regarding regeneration, BA, NAA, silver nitrate (AgNO3), and adenine hemisulfate (As) were used in ten different combinations. The ‘Cameron’, ‘Tabasco’, and ‘Noblesse’ cultivars with 95.24% regeneration percentage showed the highest rate of plant regeneration. Generally, in most cultivars, the highest regeneration rate and shoot number per explant were found in the MS medium supplemented with 3 mg·dm-3 of BA, 0.6 mg·dm-3 of NAA, 5 mg·dm-3 of AgNO3, and 40 mg·dm-3 of As. According to the results, the highest regeneration frequency was obtained when 40 mg·dm-3 of As was added to the medium. Finally, the flow cytometry analysis indicated that there were no significant differences between in vitro regenerated and control plants in terms of DNA ratios.

Regeneration potential of different explants during micropropagation of neem tree (Azadirachta indica A. Juss.)

Azadirachta indica A. Juss., (Neem), a prodigious multipurpose tree, has immense potential to benefit mankind and to protect the environment. In order to investigate the effects of three different explants for its regeneration potential, de embryonated cotyledon, immature zygotic embryo and nodal segments from a 30 year old neem plus tree were used. Half strength MS medium with benzyl amino purine (3 mg/L) and naphthalene acetic acid (0.5 mg/L) and casein hydrolysate (1 g/L) was effective in shoot bud sprouting from both nodes and cotyledons. Half strength MS medium fortified with TDZ (0.2 mg/L) was effective for induction of somatic embryogenesis from zygotic embryos. Shoot buds initiated from the cotyledons produced a maximum number of shoots per explants (4.33) which on further sub culturing induced maximum multiple shoots (15) on half strength MS medium fortified with BAP (1.5 mg/L), NAA (0.5 mg/L) and CH (400 g /L) and the nodal explants induced only 4-5 axillary shoots on further sub culturing. Even though immature zygotic embryos produced more number of somatic embryos per explant (24.97) within a short time (30-45 days), the plantlet conversion was poor (25.52 %). In vitro rooting was observed in half strength MS medium supplemented with IBA (2 mg/L). The regeneration potential of de embryonated cotyledons through a simple regeneration system may be beneficial for efficient mass propagation of selected plus trees of neem.

Effect of Different Levels of Catalysts and Chemicals Primer on Content of Induced Callus Tissue of Cotyledon Leaves of Spilanthes Acmella Seedlings on Some Chemical Traits in Vitro

This experiment was conducted in the Plant Tissue Culture Laboratory, College of Agricultural Engineering Sciences, University of Baghdad for the period from September/2018 to July 2019. The induced callus from the cotyledon leaves of seedlings of the Spilanthes acmella plant was used in order to know the effect of chemical catalysts and Starmedium was added to Glutamine (250, 300, 350) mgters on the chemical content. After 4 weeks of planting, the primary callus was planted at 150 mg in the nutrient medium supplemented with auxin,4-dichlorophenoxy acetic acid (2,4-D) 2.0 mg.L−1 and cytokinin Benzyl Adenine (BA) 0.5 mg.L−1 at constant concentrations in the first five medium, to which the catalyst was added salicylic acid at concentrations (25, 50, 75) μmol). The second medium was added to methyl jasmonate at concentrations (25, 50, 75 μmol) of the third medium was added to Casein hydrolysate at concentrations (25, 50, 75 μmol) of the fourth medium was added to Glutamine (250, 300, 350) mg. L−1. The results showed that the treatment of nutritional medium with high concentrations of stimulants and primer led to a significant increase in the content of plant tissues (the induced callus from the cotyledons) of total carbohydrates, the percentage of protein, the content of callus from the carotene pigment and content of proline, while the comparison treatment was the most effective in vegetable tissue contents of total carbohydrates and protein percentage and content of callus from the carotene pigment and proline, as well as this confirms that all treatments led to a positive and direct increase of chemical compounds content of plant tissues of chemical traits, especially in the treatment of Salicylic acid, methyl jasmonate, casein hydrolysate, glutamine, and phenylalanine (75 micromoles, 75 micromoles, 75 micromoles, 350 mg.L−1, 150 micromoles) respectively, were followed by the treatments of Salicylic acid, methyl jasmonate, casein hydrolysate, glutamine and phenylalanine (50 μmol, 50 μmol, 50 μmol, 300 mg.L−1, 100 μmol), respectively. The aim of this study is to know the effect of Salicylic acid, methyl jasmonate, casein hydrolysate, glutamine, and phenylalanine in the induction and differentiation of callus of cotyledon leaves cotyledon leaves of Spilanthes acmella seedlings on some chemical traits in vitro.

Whey Protein Isolate, Tween 20 and Casein Hydrolysate Stabilized O/W Nano-Vesicular Emulsion Systems with Curcumin Cargo

Stable oil-in-water nanoemulsions were generated by ultra-high-pressure homogenization (UHPH) at 140 and 210 MPa for use as nano-vesicular vehicles (NVV) to carry hydrophobic generally recognized as safe (GRAS) curcumin (CU) by whey protein isolate (WPI) in aqueous nutraceutical systems. Curcumin was used for its antioxidant activity and participation in the Michael reaction with nucleophiles at pHs above 8.0. Two variables, (1) addition of casein hydrolysate (CH) (2%, w/w of WPI) and, (2) use of UHPH (140 and 210 MPa), were studied for their effect on the stabilization of monodispersed NVV and antioxidant capacity of the CU as cargo in the NVV throughout storage. CH and Tween 20 both were added to increase dispersibility and stability of the NVV. Addition of CH reduced nano-particle size (dvs) by 17% at 210 MPa when compared to140 MPa (P<0.05), and increased the stability with UHPH pressure as reflected by a 63% smaller dvs at 210 MPa as compared to 140 MPa (P<0.05). The nanoparticle distribution was not changed by the addition of CU, with dvs’s of 101 and 93 nm at 140 MPa and 73 and 92 at 210 MPa for NVV and CU-NVV, respectively. The NVV system was stable for 28 days as observed in zeta-potential, contact angle, and surface energy, and can be used to deliver CU and maintain its antioxidant activity.

The effect of adding casein hydrolysate as a protein source in the culture of the bacteria paenibacillus polymyxa at wilker office of food and horticultural plants protection (PTPH) Bojonegoro

agencia(Biological Control AgentsBiological Control Agents) are currently widely used for the purpose of controlling pests and diseases or plant-disturbing organisms. One of the biological agents that has many benefits for controlling several types of diseases in both food crops and horticulture is Paenibacillus polymyxa. These bacteria are beneficial in nitrogen fixation, promotion of plant growth, solubilization of soil phosphorus, and production of exopolysaccharides, hydrolytic enzymes, antibiotics, and cytokinins. Paenibacillus polymyxa also produces polymyxin antibiotics and it is known that these bacteria contain the hormone gibberellins. Casein hydrolyzate is one of the growth media that can be used for the growth of microorganisms. The media is a complex mixture of 18 amino acids, vitamins, calcium, phosphate, and several microelements which results in their high price. Casein hydrolyzate is known to be more effective for plant tissue culture. Therefore, the purpose of this study was to determine whether the amino acid content in casein hydrolyzate could affect the growth ofbacteria Paenibacillus polymyxa. The simple medium used in this study was soybean boiled water. Soybean as an alternative medium for protein sources to substitute beef extract, beef extract and bacto peptone for the growth ofbacteria Paenibacillus polymyxa. Based on TPC calculation with graded dilution in Paenibacillus polymyxa, it was found that there was no difference in the number of bacteria to the addition of casein. This shows that bacteria can grow optimally even though casein hydrolyzate is not given during the growth process.

In vitro propagation of six selected sugarcane mutant clones through leaf explants

Six mutant clones of sugarcane with high productivity have been produced through tissue culture techniques combined with mutations using gamma-ray irradiation and Ethyl Methane Sulfonate. The six mutant clones have been tested for stability in the field. They are proven to have high productivity and yields, so that they are very potential to be developed as superior varieties. To support the planting material sufficiency of these clones, an efficient propagation method was needed. Media formulations with different physical properties and composition of growth regulators were tested to obtain high seedling propagation rates. The media formulation for callus induction was Murashige dan Skoog (MS) + 3 mg/l 2,4-D + 3 g/l casein hydrolysate + 3% sucrose and for shoot regeneration was MS + 0,5 mg/l BA + 0,1 mg/l IBA + 100 mg/l PVP and 2% sucrose. Shoot proliferation was carried out on MS liquid (1, ½) + (0.3; 0.5 mg/l) BA + 0.1 mg/l IBA + 1 mg/l Kinetin + (0; 0.5 mg/l) GA3+ sucrose 2%. The results showed that callus induction, callus regeneration, and shoot proliferation of sugarcane mutant clones were influenced by the genotype and medium composition. The fastest callus induction was obtained from the MSP-4 clone (5.82 days), and the longest was MSB-7 (8.82 days). The largest callus diameter was obtained from MSB-6 clone on MS medium containing 1 mg/l BA, 100 mg/l PVP, and 2% sucrose. The highest number of shoots was obtained from the MSB-6 clone, while the least number of shoots conducted from the MSB-8 clone. The MSB-8 clones were more difficult to regenerate compared to the others. The best media formulation for shoot proliferation was ½ MS containing 0.5 mg/l BA, 1 mg/l Kinetin, and 0.1 mg/l IBA, while the best formulation for rooting was ½ MS.

Partial-, Double-Enzymatic Dephosphorylation and EndoGluC Hydrolysis as an Original Approach to Enhancing Identification of Casein Phosphopeptides (CPPs) by Mass Spectrometry

The identification of phosphopeptides is currently a challenge when they are part of a complex matrix of peptides, such as a milk protein enzymatic hydrolysate. This challenge increases with both the number of phosphorylation sites on the phosphopeptides and their amino acid length. Here, this paper reports a four-phase strategy from an enzymatic casein hydrolysate before a mass spectrometry analysis in order to enhance the identification of phosphopeptides and phosphosites: (i) the control protein hydrolysate, (ii) a two-step enzymatic dephosphorylation of the latter, allowing for the almost total dephosphorylation of peptides, (iii) a one-step enzymatic dephosphorylation, allowing for the partial dephosphorylation of the peptides and (iv) an additional endoGluC enzymatic hydrolysis, allowing for the cleavage of long-size peptides into shorter ones. The reverse-phase high-pressure liquid chromatography–tandem mass spectrometry (RP-HPLC-MS/MS) analyses of hydrolysates that underwent this four-phase strategy allowed for the identification of 28 phosphorylation sites (90%) out of the 31 referenced in UniprotKB/Swiss-Prot (1 June 2021), compared to 17 sites (54%) without the latter. The alpha-S2 casein phosphosites, referenced by their similarity in the UniProt database, were experimentally identified, whereas pSer148, pThr166 and pSer187 from a multiphosphorylated long-size kappa-casein were not. Data are available via ProteomeXchange with identifier PXD027132.

TISSUE CULTURE MULTIPLICATION OF Paphiopedilum insigne DEPENDING ON THE MEDIUM TYPE, GROWTH REGULATORS AND NATURAL SUPPLEMENTS

Paphiopedilum is an ornamental orchid used mainly for interior decoration. As the division of plants is uneconomical, a fast method of propagation is needed. The aim of this study was to evaluate the influence of medium type (MS or VW), growth regulators, i.e. BA, KIN, TDZ used separately or in combinations and natural additives, i.e. coconut water, banana pulp, casein hydrolysate, on Paphiopedilum insigne plantlets grown in vitro. It was found out that BA in concentration of 0.5 mg·dm–3 allowed to obtain the highest multiplication rate (2.92), however the use of KIN in concentration of 1 mg·dm–3 resulted in formation of bigger and higher quality plantlets, It is possible to replace cytokinins with other biologically active substance, such as banana pulp. The 1/2 MS medium might be used for Paphiopedilum insigne tissue culture, as there was no difference in terms of multiplication rate and the obtained plantlets were of better quality, especially that it is cheaper and easier to prepare.