Percentage Of Callus Research Articles

Growth Regulator Indole-3-Butyric Acid on Rooting Potential of Actinidia deliciosa Rootstock and Actinidia arguta Female Scion Species Stem Cuttings

A study was conducted to access the effectiveness of exogenous application of indole-3-butyric acid (IBA) on adventitious root formation in kiwifruit semi-hardwood stem cuttings (SCs) from Actinidia deliciosa rootstock and Actinidia arguta female scion. Treatment comprised IBA concentrations of 0, 10, 100, 1000, 10,000 and 100,000 ppm. Parallel experiments for A. deliciosa and A. arguta’s treatment were arranged in a randomised complete block design, with 12 replications. In A. deliciosa, treatments had significant (p ≤ 0.05) effects in rooting percentage, number of roots, root length, size of callus formation and callus percentage, except for dry root mass. In A. arguta, treatments showed significant (p ≤ 0.05) effects in rooting percentage, number of roots, root length and dry root mass. No callus formation was observed in A. arguta. Relative to the control, in A. deliciosa, the highest (42%) rooting percentage and lengthy (0.301 cm) roots, were observed at 10,000 ppm IBA concentration, whereas the most (0.295) number of roots were produced at 100,000 ppm IBA concentration. Calli percentage (94%) was highest at 100 ppm IBA, while the size of callus formation was the biggest (2.8) at IBA concentration of 100,000 ppm, when compared to the control. In A. arguta, the highest (100%) rooting percentage was achieved at the control (0 ppm), 100 ppm and 10,000 ppm IBA concentrations, whereas the greatest (0.9815) number of roots were observed at the IBA concentration of 10 ppm. Lengthy (1.0839 cm) roots were achieved at IBA concentration of 100 ppm, whereas the greatest (0.1061 g) dry root mass was attained at IBA concentration of 10,000 ppm. In conclusion, the use of growth regulator IBA was effective for root formation in SCs of A. deliciosa rootstock. In A. arguta female scion, IBA application improved the quality of rooting (more and longer roots). IBA application showed its potential in stimulating root development at 10,000 ppm IBA.

Efficient regeneration protocol for producing true-to-type oil palm (Elaeis guineensis (jacq.) through somatic embryogenesis from immature male inflorescence.

Producing homogeneous planting material in oil palm poses a significant challenge, which can be addressed through somatic embryogenesis. This study successfully achieved somatic embryogenesis using immature male inflorescence from Tenera hybrid. Modified Eeuwens medium yielded better results than the Murashige and Skoog (MS) and CHU (N6) media when supplemented with 2,4-D, picloram and α-naphthaleneacetic acid (NAA). The Eeuwens (Y3) medium supplemented with picloram led to an increased callus percentage, ranging from 25% to 63.7%. The highest rate of somatic embryogenesis was observed with a combination of 2,4-D (16mg/l), picloram (24mg/l), and 2,4,5-trichlorophenoxyacetic acid (2,4,5 -T) (2mg/l), resulting in an average embryogenesis percentage between 2.54% and 7.24%. The average embryogenesis percentage varied from. Additionally, the liquid media proved effective for root growth and proliferation (R2 medium), which included Indole-3-butyric acid (IBA) (2mg/l) and NAA (0.5mg/l), along with activated charcoal (100mg/l). The elongated, nodular-shaped root-like structures developed into root primordial structures. This protocol will be instrumental in producing homogeneous oil palm planting material through micropropagation and cloning of mother palms, facilitating the establishment of semi-clonal seed gardens.

Exogenous IBA stimulatory effects on root formation of Actinidia deliciosa rootstock and Actinidia arguta male scion stem cuttings

Recalcitrance to root of kiwifruit (Actinidia spp.) stem cuttings (SCs) is a restriction for its commercial production. Exogenous hormone is the key factor to regulate root formation, but the research on exogenous hormone stimulatory effects on Actinidia spp. is still lagging. Kiwifruit plants had been mostly propagated by seeds. However, vegetative propagation offers several advantages over sexual propagation, among them being crop homogeneity, practicability, and simplicity of the technique. A study was therefore conducted to investigate the potential responses of kiwifruit SCs from Actinidia deliciosa rootstock and Actinidia arguta male scion SCs treated with indole-3-butyric acid (IBA) concentrations at low (10, 100 ppm), medium (1,000, 10,000 ppm) and high (100,000 ppm) levels. Parallel experiments for A. deliciosa and A. arguta’s treatments were arranged in a randomized complete block design, with 12 replications. Treatments comprised different IBA concentrations, namely, 0 (control), 10, 100, 1,000, 10,000 and 100,000 ppm. At 103 days, rooting percentage, number of roots, root length, dry root mass, size of callus formation and callus percentage were determined. In A. deliciosa SCs, treatments had high significant (p ≤ 0.05) effects in rooting percentage, number of roots, root length, size of callus formation and callus percentage, except for dry root mass. In A. arguta male SCs, treatments also, showed high significant (p ≤ 0.05) effects in the number of roots, root length and dry root mass, except for the rooting percentage. No callus formation was observed in A. arguta male SCs. Relative to the control, in A. deliciosa SCs, the highest (42%) rooting percentage and lengthy (0.3 cm) roots, both were observed at the medium (10,000 ppm) IBA concentration, whereas the most (0.29) number of roots were produced at the highest (100,000 ppm) IBA concentration. Calli percentage (94%) was highest at low (100 ppm) IBA, while the biggest (2.8) size of callus formation was at the highest IBA concentration of 100,000 ppm, when compared to the control. In A. arguta male SCs, the greatest number of roots (1.08) and root mass (0.07 g) were attained at the medium IBA concentration of 10,000 ppm. The longest (0.94 cm) root length was achieved at the low IBA concentration of 100 ppm. The SCs of A. arguta male scion required low to medium (100 to 10,000 ppm) concentrations of IBA for proper roots stimulation and development, whereas in SCs of A. deliciosa, highest rooting percentage paired with callus formation, was achieved at medium to higher (10,000 to 100,000 ppm) IBA concentrations. Principal component analysis (PCA) revealed that PC1 and PC2 accounted for 99% of the entire variation. While PC1 accounted for 94% of the total variation, PC2 accounted for 5% of the total variation in A. deliciosa. However, in A. arguta PCA revealed that PC1 and PC2 accounted for 93% of the entire variation. While PC1 accounted for 72% of the total variation, PC2 accounted for 21% of the total variation. The use of exogenous application of IBA was effective for adventitious root formation in SCs of A. deliciosa rootstock and A. arguta male scion. IBA showed its effectiveness in stimulating root development at 10000 ppm IBA.

Effectiveness of antioxidants on in vitro regeneration of Musa paradisiaca var. raja to prevent browning and enhance embryo development

The cultivation of raja banana is widespread in Indonesia. The process of increasing banana propagation through in vitro culture encounters a specific issue, namely the occurrence of browning during the initiation stage, which hinders the regeneration process. The objective of this study is to determine the effectivenessof antioxidants on the in vitro regeneration of raja banana to prevent browning and enhance embryo development. The study was conducted using a completely randomized design with the treatment of the antioxidant compounds ascorbic acid (AS) and melatonin (MN). The treatments consisted of control (without antioxidants), ascorbic acid (100 mg L-1, 150 mg L-1, and 200 mg L-1) and melatonin (10 mg L-1, 12 mg L-1, and 14 mg L-1). The results showed that ascorbic acid and melatonin treatment had no significant effect on the percentage of viable explants and the level of browning intensity, but provided significant results on the regeneration process. The application of melatonin at 14 mg L-1 significantly increased callus regeneration. Furthermore, at a concentration of 12 mg L-1 showed the highest callus percentage value compared to other treatments. The melatonin 12 mg L-1 treatment showed the earliest scutellar embryo formation, whereas ascorbic acid at a concentration of 100 mg L-1 resulted in the most optimal regeneration of globular and scutellar embryos. The highest concentrations of ascorbic acid and melatonin inhibit the formation of coleoptilar embryos

Comparative growth response of three rubber (Hevea brasiliensis Muell. Arg) clones in somatic embryo propagation

Abstract Plants have the ability to grow with different responses depending on the clones and varieties of plants. In rubber plants, each clone has specific characteristics or traits that distinguish it from other clones, whether in terms of growth, latex production, or resistance to pests and diseases. The objective of this experiment was to assess the response of three rubber (Hevea brasiliensis Muell. Arg) clones to the same media conditions for the growth and development of somatic embryos (SE). Integuments from three rubber clones, namely PB 260, PB 330, and PB 340, were used as explants to induce embryonic callus on MH media. After four weeks post-induction, the callus was sub-cultured to expressed SE then SE was transferred to the RITA® bio-reactor for plantlets development in liquid MD media. Parameters for each steps, such as the percentage of contamination, percentage of callus response, color of callus, percentage of SE induction, number of stage of SE, and number of shoots were recorded. The results revealed that the highest percentage of viable callus was observed in the PB 260 clone at 23.20%. On SE expression, the highest being in clone PB 340 at 2.04%. On SE development the highest shoot growth was observed in the PB 260 clone, with a total of 5 shoots.

Efficient extraction methods and callus culture of quercetin from Indian white radish: yield analysis via HPLC quantification and assessment of cytotoxic activity

SummaryIndian white radish Co. 1 (Raphanus sativus L.) contains medicinal quercetin, and its commercial value supports large‐scale quercetin isolation. Various extraction methods, including ultra‐assisted extraction (UAE), maceration extraction (ME), Soxhlet extraction (SE), conventional extraction (CSE) and serial maceration extraction (SME), were employed to extract quercetin from radish leaf sections. RP‐HPLC‐optimising key experimental parameters yielded a linear response for the individual target compounds in the concentration range, with a limit of quantification (LOQ) of 2.2 μg mL−1 (r2 = 0.9995), a limit of detection (LOD) of 2.2 μg per 1.0 g of crushed leaves and repeatability within the relative standard deviation (RSD) of 0.23–0.37%. For in vitro callus cultures, 2,4‐dichlorophenoxyacetic acid (2,4‐D), 6‐benzylaminopurine (BAP) and zeatin were used for callus induction from in vitro leaf segments of radish. MS medium enriched with 2,4‐D (0.5 mg) induced the highest percentage of callus at 4 weeks. Among the extraction methods, the SME method yielded the highest recovery of quercetin (96.9%; 1.92 mg g−1 DW), phenolic (26.07 mg g−1 DW) and flavonoid (34.03 mg g−1 DW), compared to other methods and solvents. The callus extract included 0.5 mg of 2,4‐D, 32.07 mg of phenolic, 42.03 mg of flavonoid and 6.46 mg of quercetin. The presence of phytocompounds and quercetin was confirmed by GC–MS/MS and RP‐HPLC. The isolated quercetin (57.9 μg) also showed lethal effects on HT‐29 human colon cancer cells. This method may be effective for evaluating quercetin in radish extracts.

Browning Prevention Method in Kepel (Stelechocarpus burahol (Blume) Hook.f. & Thomson) in Vitro Culture

Browning of explants is a common problem in Kepel (Stelechocarpus burahol (Blume) Hook.f. & Thomson) in vitro culture, resulting in low explant in vitro regeneration of Kepel. Using nodal explant, the effect of explant immersion in ascorbic acid, the addition of ascorbic acid to the media, and dark and light incubation conditions were investigated. The browning prevention method was selected based on the delayed browning appearance time, the lowest broning intensity, and the highest percentage of callus. This present study used a completely randomized design with treatment variations: immersion explant in ascorbic acid 100 mg/L, addition of ascorbic acid 100 and 200 mg/L to the media, addition of ascorbic acid 100 and 200 mg/L and activated charcoal 1 g/L to the media, and incubation in dark and light conditions for 28 days. The results showed that the combination of the addition of ascorbic acid 200 mg/L to MS media and incubation in dark conditions were effective browning prevention methods in inhibiting browning appearance time (5 DAP), significantly reducing browning intensity (0.3), and increasing callus growth (100%) of kepel node explants during 28 days of culture. The results of this study are useful in establishing protocols for in vitro culture of Kepel plants, especially at the initiation stage, and can be expected to support the successful conservation of Kepel plants through in vitro propagation. Keywords: Ascorbic acid, Activated Charcoal, Browning, In Vitro Culture, Stelechocarpus burahol

Induksi Kalus dari Hipokotil Belimbing Merah (Baccaurea angulata) dengan Penambahan 2,4-D (Dichlorophenoxy Acetic Acid) dan BAP (6-Benzyl Amino Purin)

Red starfruit is one of the endemic plants of Kalimantan which produces secondary metabolites which has been empirically used as antioxidant, antibacterial, and anticancer. This plant has a long reproductive period and seedless fruit is often found during the fruiting season, which is a problem for the sustainability of the red starfruit in nature. Conservation of red star fruit for propagation and production of secondary metabolites can be done in vitro through callus culture. This research aims to determine the effect of adding the growth regulators dichlorophenoxy acetic acid (2,4-D) and 6-benzyl amino purine (BAP) on callus induction time, percentage of callus forming explants, callus color and texture, and callus growth. This research used a Completely Randomized Design (CRD) with a factorial pattern consisting of two treatment factors, namely 2,4-D (0; 0.25; 0.5; 0.75; 1 ppm) and BAP (0; 0.25; 0.5; 0.75; 1 ppm). The percentage of explants forming callus (%), the time the callus appeared (days after planting), the color and texture of the callus, the wet weight and dry weight of the callus (g) were research observation parameters.The results showed that the single factor 2,4-D, single factor BAP, and the interaction of 2,4-D and BAP had a significant effect to callus appearance time. Single administration of 2,4-D and BAP had a significant effect on callus dry weight. The color variations of the callus produced are white, green and brown with a compact texture. The fastest callus emergence time was at 11.00 days after planting (DAP), namely in the combination treatment of 1 ppm 2,4-D and 0.75 ppm BAP, single treatment 0.75 ppm and 1 ppm 2,4- D. The highest average dry weight of 0.024 g was obtained in the single treatment of 0.75 ppm BAP.

<em>In Vitro</em> Effect of PEG and Proline on Callus Growth and Minerals Values in Basmati Rice (<em>Oryza sativa</em>)

Purpose: Drought is a universal issue that disturbs photosynthetic system, adversely affecting quality and crop yield. The study was conducted to determine response of polyethylene glycol (PEG) as stressing agent alone or combination with proline on four fine rice genotypes for callus components as well as biochemical parameters like Chromium (Cr), Magnesium (Mg), Manganese (Mn), Iron (Fe), Phosphorus (P).Research Method: Callus was induced on media having 4.43g/L MS + 4mg/L 2, 4-D (2, 4-dichlorophenoxy acetic acid) + 30g/L sucrose + 1.76g/L phytagel with different levels of PEG “1.25g/L and 2.5g/L” with and without 1ml/L proline.Findings: Callus was used for different analyses after 30 days and results showed that PEG treatments have a negative effect on callus fresh weight, callus dry weight, days to callus induction, callus percentage, and mineral nutrients like K, Cr, Mg, Mn, Fe, P. PEG 2.5g/L showed a greater decrease as compared to PEG 1.25g/L. Proline significantly raises callus growth alone or in combination with PEG but less than control whereas callus dry weight, K, and Ca contents increased more than control when proline applied alone or with PEG combination.Originality/value: Exogenous proline application improves callus growth rate as well as macro- micro elements by mitigating the effect of PEG.

Callus Formation and Its Characteristics of Mountain Papaya in Vitro Endosperm Culture

Callus Formation and Its Characteristics of Mountain Papaya <i>in Vitro</i> Endosperm Culture

Micropropagation from cotyledonary explants of grapefruit (&lt;i&gt;Citrus&lt;/i&gt; &lt;i&gt;paradisi&lt;/i&gt; Macf.) by indirect and direct organogenesis

Grapefruit (Citrus paradisi Macf.) is an economic fruit crop worldwide cultivated for its nutritional value, and aromatic flavours. However, the development of new varieties and high production of quality seedling constitutes a limit to improving grapefruit yields. An efficient regeneration protocol could be of great interest for grapefruit transformation, micropropagation, and germplasm conservation. In vitro plant regeneration systems of Citrus paradisi have been studied by using cotyledonary explants. Different concentrations of plant growth regulators were used to explore the organogenic potential of cotyledonary explants under laboratory conditions. For indirect organogenesis using cotyledon segment explants, a low concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) lead to the formation of white callus, while a high concentration of 2,4-D induced brown callus and adventitious root differentiation. The best response for callogenesis was obtained on Murashige and Skoog (MS) medium supplemented with 2 mg/L of 2,4-D. Only brown callus cultured on MS added with 2 mg/L 6-benzylaminopurine (BAP) has regenerated shoots, but the percentage of callus producing shoots was very low (6.66 ± 2.68%). Regarding direct organogenesis from the cotyledonary node explants, the best response for shoot proliferation was observed on MS supplemented with 4 mg/L BAP (86.66 ± 12.58% of explants regenerating shoots, and an average number of 5.45 ± 0.45 shoots per explant). Low percentages of In vitro rooting of regenerated shoots were observed with different concentrations of α-naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA). Nevertheless, the highest rooting percentage (13.33 ± 2.88%) was obtained on MS containing 2 mg/L NAA. These results provide new information for mass regeneration and the production of healthy seedlings, and can be applied in grapefruit improvement programs.

Studies on Improving the Efficiency of Somatic Embryogenesis in Grapevine (Vitis vinifera L.) and Optimising Ethyl Methanesulfonate Treatment for Mutation Induction.

Somatic embryogenesis (SE) has many applications in grapevine biotechnology including micropropagation, eradicating viral infections from infected cultivars, mass production of hypocotyl explants for micrografting, as a continuous source for haploid and doubled haploid plants, and for germplasm conservation. It is so far the only pathway for the genetic modification of grapevines through transformation. The single-cell origin of somatic embryos makes them an ideal explant for mutation breeding as the resulting mutants will be chimera-free. In the present research, two combinations of plant growth regulators and different explants from flower buds at two stages of maturity were tested in regard to the efficiency of callusing and embryo formation from the callus produced in three white grape cultivars. Also, the treatment of somatic embryos with the chemical mutagen ethyl methanesulfonate (EMS) was optimised. Medium 2339 supplemented with β-naphthoxyacetic acid (5 μM) and 6-benzylaminopurine (BAP-9.0 μM) produced significantly more calluses than medium 2337 supplemented with 2,4-dichlorophenoxyacetic acid (4.5 µM) and BAP (8.9 µM) in all explants. The calluses produced on medium 2337 were harder and more granular and produced more SEs. Although the stage of the maturity of floral bud did not have a significant effect on the callusing of the explants, calluses produced from immature floral bud explants in the premeiotic stage produced significantly more SEs than those from more mature floral buds. Overall, immature ovaries and cut floral buds exposing the cut ends of filaments, style, etc., tested for the first time in grapevine SE, produced the highest percentage of embryogenic calluses. It is much more efficient to cut the floral bud and culture than previously reported explants such as anthers, ovaries, stigmas and styles during the short flowering period when the immature flower buds are available. When the somatic embryos of the three cultivars were incubated for one hour with 0.1% EMS, their germination was reduced by 50%; an ideal treatment considered to obtain a high frequency of mutations for screening. Our research findings will facilitate more efficient SE induction in grapevines and inducing mutations for improving individual traits without altering the genetic background of the cultivar.

REGENERASI TANAMAN ANGGREK MERPATI (Dendrobium crumenatum Swartz) PADA MEDIA KULTUR DENGAN TAMBAHAN ZEATIN DAN SUKROSA

The need for orchids is increasing and the orchids are threatened with extinction due to forest exploitation and need to be supported by the provision of seeds using tissue culture technology. The research entitled regeneration of the pigeon orchid (Dendrobium crumenatum Swartz) aim of this study was to determine the effect of the interaction between the concentrations of growth regulators zeatin and sucrose on the growth of Dendrobium crumenatum Swartz callus in vitro. The experimental design used was a factorial randomized block design consisting of 2 factors. The first factor is the zeatin dose consisting of without treatment, 0.1, 1.0, and 10 ppm. The second factor is the sucrose dose consisting without treatment, 20, 40, and 60 g/l, there are 16 treatment combinations and 3 repetitions so that 48 experimental units were obtained. Each experimental unit consisted of 4 explants, so that a total of 192 explants were explants. The parameters observed were as follows: percentage of live explants, percentage of rooted explants, percentage of explants that formed callus, number of shoots, number of leaves, and chlorophyll content Follow-up test Honest Significant Difference (HSD) at 5% level. The interaction of zeatin and sucrose affects the survival percentage of explants and callus, the number of shoots and the number of leaves. The best treatment was 1.0 ppm zeatin and 40 g/l sucrose. The main effect of the best zeatin concentration is 1.0 ppm and the best dose concentration is sucrose 40 g/l.

Effect of the Combination of the Growth Regulators and Putrescine on the Somatic Embryogenesis of Wheat (Triticum Aestivum L.) on Some Types of Explants

Plant breeding programs need to be carried out in order to improve the genetics of wheat that is able to adapt to tropical environments through hybridization, mutation induction, tissue culture, and genetic transformation. In vitro culture through somatic embryogenesis pathways plays an important role in genetic improvement and its integration with other breeding programs can positively affect the improvement of wheat quality, quantity, and development in Indonesia. The purpose of this study was to obtain an embryogenic callus induction method from the Dewata variety using five different types of explants, namely mature Seeds, immature embryos, immature seeds, leaf, stem, and to obtain combination of plant growth regulators and putrescine on somatic embryogenesis of wheat. The experimental design was prepared based on a complete randomized design with a combination treatment of embryogenic callus induction media consisting of 9 levels, namely: 1 ppm 2.4- D, 1 ppm 2.4-D + 1 ppm Picloram, ppm 2.4-D + 1 mg / L Picloram, 1 ppm 2.4-D + 10-4 M Putrescine, 1 ppm 2.4-D + 1 ppm Picloram +10-4 M Putrescine, 2 ppm 2.4-D + 1 ppm Picloram + 10-4 M Putrescine, 1 ppm 2.4 D + 10-3 M Putrescine, 1 ppm 2.4 D + 1 ppm Picloram + 10-3 M Putrescine, 2 ppm 2.4 D + 1 ppm Picloram + 10-3 M Putrescine. The results showed that the media used was able to induce embryogenic callus using mature seed and immature embryo, but immature seed and leaf were not able to produce embryogenic callus. The best media that produced the highest percentage of embryogenic callus was 2 ppm 2.4-D + 1 ppm Picloram + 10-4 M Putrescine with as much as 85.9% in young embryo explants.

Modeling Callus Induction and Regeneration in Hypocotyl Explant of Fodder Pea (Pisum sativum var. arvense L.) Using Machine Learning Algorithm Method

A comprehensive understanding of genetic diversity and the categorization of germplasm is important to effectively identify appropriate parental candidates for the goal of breeding. It is necessary to have a technique of tissue culture that is both effective and reproducible to perform genetic engineering on fodder pea genotypes (Pisum sativum var. arvense L.). In this investigation, the genetic diversity of forty-two fodder pea genotypes was assessed based on their ability of callus induction (CI), the percentage of embryogenic callus by explant number (ECNEP), the percentage of responding embryogenic calluses by explant number (RECNEP), the number of somatic embryogenesis (NSE), the number of responding somatic embryogenesis (RSE), the regeneration efficiency (RE), and the number of regenerated plantlets (NRP). The findings of the ANOVA showed that there were significant differences (p &lt; 0.001) between the genotypes for all in vitro parameters. The method of principal component analysis (PCA) was used to study the correlations that exist between the factors associated with tissue culture. While RE and NRP variables were most strongly associated with Doğruyol, Ovaçevirme-4, Doşeli-1, Yolgeçmez, and Incili-3 genotypes, RECNEP, NSE, RDE, and RECNEP variables were strongly associated with Avcılar, Ovaçevirme-3, and Ardahan Merkez-2 genotypes. The in vitro process is a complex multivariate process and more robust analyses are needed for linear and nonlinear parameters. Within the scope of this study, artificial neural network (ANN), random forest (RF), and multivariate adaptive regression spline (MARS) algorithms were used for RE estimation, and these algorithms were also compared. The results that we acquired from our research led us to the conclusion that the employed ANN-multilayer perceptron (ANN-MLP) model (R2 = 0.941) performs better than the RF model (R2 = 0.754) and the MARS model (R2 = 0.214). Despite this, it has been shown that the RF model is capable of accurately predicting RE in the early stages of the in vitro process. The current work is an inquiry regarding the use of RF, MARS, and ANN models in plant tissue culture, and it indicates the possibilities of application in a variety of economically important fodder peas.

The Effect of BAP and NAA Combination on Callus Induction of Aglaonema Siam Aurora Leaf Explants in Vitro

This research aims to determine the effect of the Naphthalene Acetic Acid and Benzyl Amino Purine combination on callus growth of Aglaonema Siam Aurora leaf explants and to determine the combination level at the most optimal concentration on the callus growth of Aglaonema Siam Aurora leaf explants. Observation variables included callus emergence time, explants percentage of formed callus, callus length, and explant life percentage. The explants that where used were the first young leaves explants from shoots that were opened, with 2x2 cut size. Results show that the addition of a NAA and BAP combination had an effect on . Combination of 1.2 ppm BAP + 1 ppm NAA treatment is the most optimum treatment for callus growth with a callus percentage of 87.5%, the callus start time at week 3, the average callus length is 0.77 mm and the percentage number of survived explants is 100%

Engineering of insecticidal hybrid gene into potato chloroplast genome exhibits promising control of Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The potato chloroplast was transformed with codon optimized synthetic hybrid cry gene (SN19) to mitigate crop losses by Colorado potato beetle (CPB). The bombarded explants (leaves and internode) were cultured on MS medium supplemented with BAP (2.0mg/l), NAA (0.2mg/l), TDZ (2.0mg/l) and GA3 (0.1mg/l); spectinomycin 50mg/l was used as a selection agent in the medium. Leaf explants of cultivar Kuroda induced highest percentage (92%) of callus where cultivar Santae produced the highest percentage (85.7%) of transplastomic shoots. Sante and Challenger showed 9.6% shoot regeneration efficiency followed by cultivar Simply Red (8.8%). PCR amplification yielded 16 postive transplastomic plantlets out of 21 spectinomycin resistant ones. Target gene integration was confirmed by PCR and Southern blot, whereas RT-qPCR was used to assess the expression level of transgene. The localization of visual marker gene gfp was tracked by laser scanning confocal microscopy which confirmed its expression in chloroplasts of leaf cells. The transplastomic plants ensured high mortality to both larvae and adult CPB. Foliage consumption and weight gain of CPB fed on transplastomic leaves were lower compared to the control plants. Sucessful implementation of current research findings can lead to a viable solution to CPB mediated potato losses globally.

Obtaining dihaploid lines of soft winter wheat with complex resistance to rust and hard smut by anther culture in vitro

Aim. Obtaining homozygous linear material of winter soft wheat of different genetic origin (complex hybrids), which are resistant to different types of rust by androgenesis in vitro. Methods. In vitro culture of isolated anthers of wheat. The percentage of callus and regeneration of green plants for each genotype calculated as a percentage of the planted anthers. Methods of phytopathological assessment of resistance to complex of various diseases. Results. The differences in the frequency of сallus induction (from 0.99 to 16.26 % of planted anthers) and the ability to regenerate plants (from 0 to 3.05 % of planted anthers) in the process of androgenesiss in vitro of winter soft wheat were detected. Fertile regenerants in the anthers culture 5 out of 9 studied hybrids have been obtained. The effectiveness of the last process was different: from 12.5 to 60.0 % (an average of 29.41 %) from the received regenerants. The high degree (8–9 points) of the resistance of the obtained dihaploids to a complex of diseases was shown. Conclusions. Genotype-specific of microspores morphogenetic reactions of soft winter wheat in the process of androgenesis in vitro were revealed. The highest level of callus formation were shown for samples 120/20 and 132/20. 15 dihaploid lines with complex rust resistance and hard smut were obtained.

Callogenesis of Dayak Onion (Eleutherine palmifolia) Bulb in response of Picloram, 2,4-D, and Kinetin

Dayak onion contains bioactive compounds that are traditionally used for medicine. The production of bioactive compounds can be increased through callus culture. This study aims to analyze the effect of the combination of plant growth regulator on the growth of Dayak bulb callus. The design of this study used a completely randomized factorial design with 2 factors. The first factor is the concentration of auxin. While the second factor is the concentration of kinetin. The parameters observed include the percentage of callus, the time of callus formation, fresh weight, dry weight, and callus morphology. In this study, a callus growth curve was also made to determine the best harvest time. The results showed that highest callus fresh weight was obtained in the P3K1 treatment. The percentage of callus formation in the picloram + kinetin treatment was higher than in the 2,4-D + kinetin treatment. The time of callus formation occurred in the combination of picloram + kinetin faster than the 2.4-D + kinetin treatment. The callus color is diverse, while the texture of the entire callus is compact. The best callus induction medium for Dayak onion bulbs is to use picloram 2-4 ppm + kinetin0.025-0.5 ppm. The results of this study provide optimal growth regulatory information for the induction of dayak onion callus that has never been reported before. This information can be the basis for the development of methods of production of bioactive compounds from dayak onions through callus culture.

In vitro Regeneration of an Endangered Medicinal Orchid Dendrobium crepidatum Lindl. &amp; Paxton through Protocorm like bodies.

A protocol for regeneration of an endangered medicinal orchid Dendrobium crepidatum was established mainly via indirect protocorm like body (PLBs) formation. The PLBs procured from in vitro growth cultures was assessed in ½ MS medium alone and in combinations with 6-Benzyl amino purine (BAP), Kinetin (Kn) and α-Naphthalene acetic acid (NAA). The calli were induced from longitudinally bisected segments of PLBs. The optimum percentage of callus was obtained in ½ MS medium supplemented with BAP (1.0 mg/l) and combination of BAP (1.0 mg/l) with NAA (0.5 mg/l), where 64.3% and 83.3% of callus was formed respectively. PLB formation was achieved when callus was transferred onto the respective combinations in ½ MS basal medium. The optimum percentage (91.6%) of PLBs were regenerated in the combination of BAP with NAA. These PLBs differentiated into intact plantlets about 3 cm in height and with 4-5 roots were transplanted into potting mixture with highest rate of 93.3% survival rate in a green house.