Murashige & Skoog Research Articles

In Vitro Multiplication of Nepenthes mirabilis Lour (Druce) with Variations Concentration of Sucrose and BAP

Nepenthes mirabilis (Lour) Druce is a rare ornamental plant and has high economic value so it needs to be preserved. This study aimed to analyze effect of sucrose and BAP concentrations and their optimal interactions in the in vitro multiplication of Nepenthes mirabilis. Explants in the form of in vitro plantlets from Laboratory of Plant Tissue Culture, Department of Biology, Semarang State University. The treatment was carried out by adding sucrose and BAP to murashige skoog (MS) media with various concentrations. The treatments were arranged in two factor completely randomized design with four replications. Plantlets were planted in the treatment medium for 10 weeks, then every week the growth of shoots and leaves of N. mirabilis was observed. The data were analyzed by two-way analysis of variance and Duncan's test. The results showed that concentration of sucrose, BAP, and their interactions affected the growth of explants. Plantlets grown on media with sucrose concentration 20 g/l produced the highest number of shoots, media with concentrations BAP 3 and 2 ppm produced the highest number of shoots and number of leaves. Medium ½ MS with varying concentrations of sucrose 20 g/l with 3 ppm BAP and sucrose 20 g/l with 2 ppm BAP resulted in the highest growth in the number of shoots and number of leaves. The composition of this medium can be used for the multiplication of N. mirabilis.

Perbanyakan Vanili (Vanilla Planifolia Andrews.) Dengan Penambahan Kinetin Melalui Teknik Kultur Jaringan Efek

The development of vanilla seeds through tissue culture techniques with the addition of growth regulator kinetin aims to increase the success of shoot formation and growth of vanilla explants. The research was carried out from February to June 2021 at the Tissue Culture Laboratory Politeknik Negeri Jember. The research method used a single factor Completely Randomized Design (CRD) with 4 treatments which were repeated 5 times. The treatment was the addition of kinetin to Murashige-Skoog (MS) media with a concentration of 0, 1, 2, 3 mg/L. Data analysis used analysis of variance followed by a 5% or 1% BNT test. The results of the analysis showed that the addition of kinetin affected the initial germination time of vanilla explants with the fastest time being 13-17 days after inoculation at a kinetin concentration of 1-3 mg/L. The kinetin concentration of 3 mg/L also affected the wet weight of vanilla explants aged 8 weeks after inoculation with the highest average of 2.62 grams per explant. However, the addition of kinetin did not affect root length, root number and shoot length until the vanilla explants were 8 weeks old after inoculation. .

Application of temporary immersion system RITA® for efficient biomass multiplication and production of artificial seeds for ex situ conservation of Linnaea borealis L

Linnaea borealis L., belonging to the Linnaeaceae family, in some European countries including Poland, is a protected species. Phytochemical studies indicate the presence of pharmacologically important secondary metabolites, which may indicate undiscovered biological activity and untapped therapeutic potential of this species. Harvesting the raw material from natural sites is impossible due to the protection status of this glacial relict in many countries. The aim of the current work was to develop an efficient protocol of Linnaea borealis L. var. borealis shoot multiplication via the development of axillary buds in an agitated culture and a temporary-immersion system as well as to establish the production of artificial seeds via alginate encapsulated nodes for short-time storage. The highest results of growth parameters were obtained for shoots agitated in MS (Murashige & Skoog) liquid medium enriched with cytokinin, auxin and gibberellin (18.3 ± 0.4 shoots per explant and 2185 ± 98% biomass increase, respectively). The growth of shoots in the RITA® bioreactor was highly efficient, especially when hormone supplementation in MS medium was used, the amount of medium in the culture vessel was 100 or 150 ml, and the immersion time of the shoots was 1 or 2 min. The survival and recovery rates of L. borealis artificial seeds, regardless of encapsulation matrix used, subsequently decreased with the increased storage duration up to 6 months (from 100 to 60% at 4 °C and from 100 to 54% at -18 ºC, respectively). This is the first report on twinflower shoot multiplication in a temporary-immersion system and artificial seeds production of this species.

Assessing the genetic fidelity of somatic embryo-derived plantlets of finger millet by random amplified polymorphic DNA analysis.

Finger millet [Eleusine coracana (L.) Gaertn.] is an important cereal because of its mineral-nutrition value. With the increasing demand, there is a pressing need to conserve it through biotechnological approaches. High-frequency somatic embryogenesis from seed-derived callus of E. coracana was developed on Murashige-Skoog (MS) medium supplemented with a combination of auxins [Indole-3-acetic acid (IAA), 2,4-Dichlorophenoxy acetic acid (2,4-D)] and cytokinins [6-Benzylaminopurine (BAP), kinetin (KN)] in different concentrations, ranging from 0.1 to 5.0mg L-1. Seeds cultured on this medium produced three different types of primary callus. Type I callus was very compact and dark brown, type II callus was light brownish and type III callus appeared whitish and light brown. All three types of calli had differential proliferation responses. Type II compact brown calli were obtained on the MS medium supplemented with 1.0 and 1.5mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5mg kinetin L-1. Friable yellowish embryogenic calli with a large number of somatic embryos were developed within 60days after being transferred to auxins and cytokinin (1.0 and 1.5mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5mg Kinetin L-1) along with 200mg casein hydrolysate L-1. Germination of somatic embryos on a half-strength MS medium supplemented with 0.1% Kinetin led to the development of healthy plantlets within 30days. Genetic fingerprinting using random amplified polymorphic DNA (RAPD) revealed high levels of genetic fidelity. The study provides methods and hormonal concentrations required to develop somatic embryos in E. coracana for its genetic improvement and conservation.

Endophy Bacterial Phenoty of Aloe Vera (Aloe barbadensis miller) as the Producer of Antibacterial Compounds Towards Eschericia coli and Staphylococcus aureus

The aims of this research are to isolate endophytic bacteria in aloe vera, to determine the antibacterial ability of endophytic bacteria contained in aloe vera against the growth of Staphylococcus aureus and Escherichia coli bacteria and to determine the characterization of endophytic bacteria found in aloe vera. This research is a laboratory experimental study with research procedures, namely sampling by purposive sampling method, isolation of endophytic bacteria carried out by strake plate, antibacterial potential test against Staphylococcus aureus and Escherichia coli using paper disc diffusion method, Morphological identification was carried out by staining bacteria gram, Phenotypic characterization of endophytic bacteria using Profile matching method. The results showed that endophytic bacteria found in aloe vera leaves were characterized by the growth of bacterial colonies on Murashige-Skoog (MS) media with different shapes and colors, the results of the antibacterial potential of endophytes against the growth of Escherichia coli and Staphylococcus aureus showed that there were 6 bacterial isolates that showed clear zones in Escherichia coli and Staphylococcus aureus bacteria and characterization results showed that isolates H2L, H9L and H10L had similar genera to Staphylococcus and Bacillus while isolates H4L, H5L and H7L had similar genera to Pseudomonas.

Влияние кверцетина на регенерацию микрорастений картофеля в условиях in vitro

Potato biotechnology in vitro has found wide application in the primary seed production of this crop - in the technologies of rehabilitation, micropropagation, depositing varietal collections in vitro, obtaining microtuber planting material. The article considers the effect of quercetin on the growth and development of potato micro plants under in vitro conditions. The objectives of the study included the morphological assessment of potato regenerants on nutrient media of various mineral compositions, as well as the study of the response of potato mericlons to the presence of quercetin in the nutrient medium. Potato varieties "Rivera" were used as objects. Murashige-Skoog (MS) Hamburg (½B5) culture media were chosen for the study. Quercetin (100 mg/l) was used as a growth stimulant. The use of a phenolic compound showed a different reaction of plants depending on the mineral composition of the nutrient medium. The presence of bioflavonoid in the MS nutrient medium contributed to a decrease in plant height, as well as the length of roots in experimental plants. Propagation of explants on nutrient medium B5 with the introduction of quercin made it possible to achieve a powerful root system. The number of first-order roots and their length increased relative to the control by 12 and 5%. The use of querticin led to a decrease in the height of the shoot due to the shortening of the internodes. Keywords: POTATOES, MICROCLONAL REPRODUCTION, BIOFLAVANOID, QUERCETIN

Re-introduction of an extinct population of Pulsatilla patens using different propagation techniques

The study focuses on the propagation of a rare and endangered plant species (Pulsatilla patens) to re-introduce an extinct population from calamine area in Southern Poland. The plants were propagated from seeds, rhizome cuttings, or regenerated in vitro from shoot tips, hypocotyls with roots or cotyledons of seedlings on Murashige & Skoog (MS) medium supplemented with 0.25 or 0.50 mg L−1 BAP (Benzylaminopurine) via direct and indirect organogenesis or somatic embryogenesis (SE). The most efficient micropropagation method was with shoot tips as an explant on MS + 0.25 mg L−1 BAP where 97% of the explants produced multiple shoots, mass SE was observed after transfer on ½ MS with 2% saccharose; 267 (35%) shoots rooted on ½ MS + 2% saccharose were acclimatized to ex vitro conditions. Flow cytometry revealed genome size stability of propagated plantlets. Low genetic differentiation between micropropagated plantlets and initial material was indicated by ISSR (Inter Simple Sequence Repeat) markers. Totally, 132 vigorous plantlets obtained on various pathways were introduced to the field plots in 2020; 30.33% survived the winter, and several reached the generative stage and flowered in the spring 2021. In next season (March/April 2022) the number of introduced plants decreased to 25% while the number of flowering and fruiting shoots in different clumps increased in some plots. This is the first report of successful re-introduction of the endangered P. patens based on micropropagation, rhizome cuttings, and seed germination.

The Effect of Media Types and NAA Concentrations on Agarwood (Aquilaria malaccensis Lamk.) Shoot Development in In Vitro Culture

<p>Agarwood (Aquilaria malaccensis Lamk.) is a forest product commodity with high economic value, but its availability in nature is decreasing due to deforestation and overexploitation. The availability of high-quality seedlings is necessary to meet the demand and conserve the sources of agarwood plants. Therefore, this study investigates the interaction effect between media types and 1-Naphthaleneacetic acid (NAA) concentrations on agarwood shoot development. It also aims to determine the best medium type and NAA concentration to stimulate agarwood shoot development. This research had two stages, namely the shoot induction and development. The shoot induction stage was conducted experimentally, using a completely randomized design (CRD) on a factorial treatment pattern of 2 factors i.e., the types of media and NAA concentrations. The shoots produced from the induction stage were cultured on Murashige-Skoog (MS) media with the same media types but without any NAA addition. Furthermore, the variables observed in both stages were the development of agarwood shoots, and the parameters measured included the number of shoots, number of leaves and shoot lengths. The results showed that the development of agarwood shoots was controlled by the type of medium used. Liquid MS medium supported with filter paper bridge produced the highest average number of shoots with 4.93 and 5.87 shoots explants-1 and length at 3.25 and 3.64 cm explant-1 in the induction and development stage, respectively. These findings will facilitate mass propagation of agarwood shoots, and in turn the availability of agarwood plantlets supports its conservation and production.</p>

Pemberian air rebusan kentang pada media Murashige-Skoog terhadap pertumbuhan protokorm anggrek sendu (Grammatophyllum stapeliiflorum) secara in vitro

Grammatophyllum stapeliiflorum is one of the most popular orchid species because of its beauty and is characterized by the shape of the flower stalk that sticks down and the brown flowers with white and yellow spots that look like drops of water or sad. This type of orchid is almost extinct and is rarely found in its natural habitat, so it is necessary to perform in vitro propagation of orchids. The culture in vitro method is one of the most effective methods for propagating rare orchids. This study aimed to determine the effect of adding potato water on protocorm growth and determine the best concentration for the growth of G. stapeliiflorum protocorm in vitro. This study used a Completely Randomized Design (CRD) with the addition of potato water (concentrations 0, 50, 100, 150, and 200 ml/L) on Murashige-Skoog (MS) medium. The results showed that the percentage of live explants was 100% in all treatments. The addition of potato water to MS medium had no effect on the percentage of live explants but still affected the growth percentage of Protocorm-like bodies (PLBs) in vitro. The best concentration of potato water for the protocorm growth of G. stapeliiflorum in vitro to MS medium was at a concentration of 150 ml/L, which was indicated by the highest percentage of PLBs growth at 100% and the color of PLBs green (2.5GY 6/8) compared to other potato water treatments and controls.

Improving Agarwood (Aquilaria malaccensis Lamk.) Plantlet Formation Using Various Types and Concentrations of Auxins

<p><em><span lang="EN-US">Aquilaria malaccensis</span></em><span lang="EN-US"> Lamk</span><span lang="EN-US">.</span><span lang="EN-US"> is one of the most </span><span lang="EN-US">widespread</span><span lang="EN-US"> agarwood-producing plants </span><span lang="EN-US">that</span><span lang="EN-US"> face extinction due to overexploitation. Agarwood propagation using <em>in vitro </em>culture techniques is capable of producing large quantities of plants in a shorter time and free from pests and diseases. </span><span lang="EN-US">Therefore, this </span><span lang="EN-US">study</span><span lang="EN-US"> was conducted to analyze</span><span lang="EN-US"> the effect of auxins type and concentration on agarwood plantlet formation using a split-plot design. The main plot was the type of auxin which included IAA, IBA and NAA</span><span lang="EN-US">, while the</span><span lang="EN-US"> subplot was the concentration used which consisted of 0; 5; 10; 15 and 20 µM. The variable observed was agarwood plantlet formation with parameters measured including the number of shoots</span><span lang="EN-US"> and</span><span lang="EN-US"> leaves, plant height, and number of roots. The results showed that the formation of agarwood plantlets was controlled by the type, concentration, and interaction between the type and concentration of auxin. Furthermore, explants cultured on Murashige Skoog (MS) medium supplemented with 10 µM IBA produced the highest number of shoots (3.39 shoots explant<sup>-1</sup>) and leaves (7.25 leaves explants<sup>-1</sup>), while the addition of 10 uM NAA resulted in the highest number of roots (2.52 roots explant<sup>-1</sup>). This is the first time a study is conducted to evaluate the effect of type and concentration of auxins on agarwood plantlet formation</span><span lang="EN-US">.</span><span lang="EN-US"> The production of high-quality shoots and plantlets increased agarwood germplasm availability to prevent extinction and support sustainable production.</span></p>

Engineered Nanomaterial Exposure Affects Organelle Genetic Material Replication in Arabidopsis thaliana.

Mitochondria and chloroplasts not only are cellular energy sources but also have important regulatory and developmental roles in cell function. CeO2, FeOx ENMs, ZnS, CdS QDs, and relative metal salts were utilized in Murashige-Skoog (MS) synthetic growth medium at different concentrations (80-500 mg L-1) and times of exposures (0-20 days). Analysis of physiological and molecular response of A. thaliana chloroplasts and mitochondrion demonstrates that ENMs increase or decrease functionality and organelle genome replication. Exposure to nanoscale CeO2 and FeOx causes an 81-105% increase in biomass, whereas ZnS and CdS QDs yielded neutral or a 59% decrease in growth, respectively. Differential effects between ENMs and their corresponding metal salts highlight nanoscale-specific response pathways, which include energy production and oxidative stress response. Differences may be ascribed to ENM and the metal salt dissolution rate and the toxicity of the metal ion, which suggests eventual biotransformation processes occurring within the plant. With regard to specific effects on plastid (pt) and mitochondrial (mt) DNA, CdS QD exposure triggered potential variations at the substoichiometric level in the two organellar genomes, while nanoscale FeOx and ZnS QDs caused a 1- to 3-fold increase in ptDNA and mtDNA copy numbers. Nanoparticle CeO2 exposure did not affect ptDNA and mtDNA stoichiometry. These findings suggest that modification in stoichiometry is a potential morpho-functional adaptive response to ENM exposure, triggered by modifications of bioenergetic redox balance, which leads to reducing the photosynthesis or cellular respiration rate.

The effect of 2,4-dichlorophenoxyacetic acid, benzyl amino purin and cupric sulphate on in vitro propagation system from shoot apices of shoot tiller of hybrid Napier grass (Pennisetum purpureum Schum)

An efficient micropropagation method of hybrid Napier grass (Pennisetum purpureumn Schum) for in vitro plant production and material breeding was established from multiple-shoot clumps (MCS) regeneration system. This system was important for forage breeding system. Shoot apices from shoot-tillers produced MSC on Murashige-Skoog (MS) induction medium containing several combinations of BAP and 2,4-D in induction stage. The addition of 5 μM (v/v) and 50 μM (v/v) CuSO4 were added in best medium for inoculation to proliferate the clump in proliferation/multiplication stage. Plant regeneration was achieved by culturing on solid MS with several combination of medium containing NAA and BAP in regeneration stage. The best results for induction were Murashige-Skoog (MS) induction medium containing 2 mgL−1 BAP and 0.1 mgL−1 2,4-D. The proliferation stage on MS medium containing 5 μM CuSÜ4 effective for proliferation (50% multiple shoot formation). The regeneration stage using 0.1 mgL−1 NAA and 2.0 mgL−1 BAP (51.6% number of shoot can regenerate). All plantlets were successfully grown up in an acclimatization stage. Based on the results, the hybrid Napier grass regeneration via MSC was a stable tissue culture system (no albino plats), which could be applied either for further genetic transformation assay or for alternative supply of nursery plant in the future.

In Vitro-Propagation of Agave tequilana Weber cv. azul in a Temporary Immersion System

In Mexico, there is a need to produce large quantities of plantlets for the establishment and replanting of blue (cv. azul) agave production areas. Most of these plots are within the origin denomination area (DOT, Spanish acronym) of the distilled product of this plant, known as tequila. The objective of this study was to develop an in vitro-propagation protocol for Agave tequilana Weber cv. azul using segmented stems in both: solid and liquid media. A disinfection and in vitro technique were developed to obtain shoots, through plantlets collected in commercial plots, which attained 100% surface-disinfection and budding rate. At the multiplication stage, the effects of 6-Benzylaminopurine (BA) (0.0, 4.4 and 13.2 μM) and kinetin (0.0, 9.4, 18.8 and 37.6 μM) were evaluated on lateral-shoot production of segmented sagittal stems. These were cultivated on Murashige & Skoog (MS) medium, with the addition of 3.0% sucrose and 8 g L−1 agar. It was observed that BA and kinetin increased the number of shoots per explant, obtaining up to 18 and 26, respectively. Furthermore, it was found that just the sagittal segmentation of explants increased axillary budding. On the other hand, segmented-stem bases were grown in MS liquid medium with 3.0% sucrose, inside a RITA® system, programmed by a 5 min immersion step with a frequency of every 4 h. The effect of Indole−3-Acetic acid (IAA) (0.57, 2.9, 5.7 μM) was evaluated, while maintaining a concentration of BA (13.2 μM). It was observed that the greatest concentration of IAA led to the formation of more than 20 buds per explant. These results offer a new methodology to increase the efficiency of A. tequilana Weber cv. azul-in vitro multiplication by sagittal segmentation of stems and the addition of BA and/or IAA.

<p><strong><em>In vitro </em>florigenesis: an efficient regeneration system avoiding time consuming vegetative phase in popular Indian soybean variety JS-335</strong></p>

Soybean ( Glycine max L.) is a major legume crop cultivated principally as protein rich bean. Improving quality and yield received considerable attention from researchers. Here, we explored molecular approaches to improve soybean quality and yield. In spite of recalcitrance, soybean plants successfully regenerated through complex and time consuming in vitro regeneration protocols via organogenesis and/or somatic embryogenesis and being used for transgenic development. Transformation efficiency is highly dependent on the regeneration as not all the cells transformed lead to the recovery of viable plant regeneration. Consequently, efficient in vitro regeneration found to be directly associated with the recovery of transformants. In the present study, we standardized the in vitro florigenesis using cotyledonary node with axillary bud as explant of soybean variety JS-335. Flower buds were directly induced from proximal end of the explant on Murashige-Skoog (MS) medium augmented with thidiazuron (TDZ) and α naphthalene acetic acid (NAA). TDZ proved a potential growth regulator to induce in vitro florigenesis. As a result of in vitro fertilization, pods were developed from flowers and matured within 40-45 days on hormone-free medium. Pods and seed set under in vitro conditions resemble pods and seeds produced under in vivo conditions. This pathway of in vitro florigenesis showed great potential for successful induction of in vitro flowering, which in turn can be explored in producing transgenic soybean seeds in popular Indian soybean genotype without escaping transgene.

Efficient in vitro plant regeneration from leaf-derived callus and genetic fidelity assessment of an endemic medicinal plant Ranunculus wallichianus Wight & Arnn by using RAPD and ISSR markers

Ranunculus wallichianus is a medicinally important plant and an endemic species to the Western Ghats of South India. An efficient and reliable indirect regeneration protocol system for R. wallichianus was developed from leaf explants in the present investigation. Leaf explants were cultured on both full-strength and half-strength MS (Murashige & Skoog) medium supplemented with different concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of 2,4-D and NAA. Among the different concentrations tested, the highest percentage of yellowish-green compact nodular callus formation was observed on a half-strength MS medium with 2.0 mg L− 1 of 2, 4-D. Then, the in vitro raised organogenic callus was cultured on a half-strength MS medium containing various concentrations (0.5 mg L− 1 to 3.0 mg L− 1) of BA, KIN, and TDZ with 0.5 mg L− 1 NAA and 10 % CW for in vitro shoot regeneration. The highest percentage of regeneration response (97 %) and a maximum number of shoots formation (11.1 ± 0.13 shoots/culture with 9.2 ± 0.21 cm mean shoot length) were obtained from MS medium containing 2.5 mg L− 1 BA with 0.5 mg L− 1 NAA and 10 % CW. The well elongated in vitro raised shoots were effectively rooted in a half-strength MS medium with 2.5 mg L− 1 IBA + 250 mg L− 1 activated charcoal. The well-rooted plantlets were successfully hardened and acclimatized with a survival rate of 94 %. Clonal fidelity of in vitro raised plantlets was assessed by using DNA-based RAPD and ISSR molecular markers. A total of 56 and 47 monomorphic bands were obtained from RAPD and ISSR markers respectively. This present in vitro propagation protocol system could be effective for the conservation of R. wallichianus with their genetic purity and its further investigations.

Protocol development for somatic embryogenesis, SSR markers and genetic modification of Stipagrostis pennata (Trin.) De Winter

BackgroundStipagrostis pennata (Trin.) De Winter is an important species for fixing sand in shifting and semi-fixed sandy lands, for grazing, and potentially as a source of lignocellulose fibres for pulp and paper industry. The seeds have low viability, which limits uses for revegetation. Somatic embryogenesis offers an alternative method for obtaining large numbers of plants from limited seed sources.ResultsA protocol for plant regeneration from somatic embryos of S. pennata was developed. Somatic embryogenesis was induced on Murashige & Skoog (MS) medium supplemented with 3 mg·L–1 2,4-D subsequently shoots were induced on MS medium and supplemented with 5 mg·L–1 zeatin riboside. The highest shoots induction was obtained when embryogenic callus derived from mature embryos (96%) in combination with MS filter-sterilized medium was used from Khuzestan location. The genetic stability of regenerated plants was analysed using ten simple sequence repeats (SSR) markers from S. pennata which showed no somaclonal variation in regenerated plants from somatic embryos of S. pennata. The regenerated plants of S. pennata showed genetic stability without any somaclonal variation for the four pairs of primers that gave the expected amplicon sizes. This data seems very reliable as three of the PCR products belonged to the coding region of the genome.Furthermore, stable expression of GUS was obtained after Agrobacterium-mediated transformation using a super binary vector carried by a bacterial strain LBA4404.ConclusionTo our knowledge, the current work is the first attempt to develop an in vitro protocol for somatic embryogenesis including the SSR marker analyses of regenerated plants, and Agrobacterium-mediated transformation of S. pennata that can be used for its large-scale production for commercial purposes.

Respon Pertumbuhan Hipokotil Kurma (Phoenix dactylifera L.) cv. Barari Madu pada Beberapa Konsentrasi Zat Pengatur Tumbuh secara In Vitro

This study aims to determine the growth response of the hypocotyl date palm (Phoenix dactylifera L.) cv. Barari on several types and concentrations of Growth Regulators (PGR) in vitro. The research was carried out by planting 30-day-old hypocotyl dates on Murashige Skoog (MS) media by giving several concentrations of ZPT Naphthalene Acetic Acid (NAA) and Benzyl Amino Purine (BAP). The treatment given is; P0 (Media MS0, without the addition of PGR), P1 (Media MS+1 ppm NAA+1 ppm BAP), P2 (Media MS+2 ppm NAA+1 ppm BAP), and P3 (Media MS+3 ppm NAA+1 ppm BAP). Each treatment consisted of 20 explants with 3 replications. The parameters observed were: 1) the first time shoots/roots appeared; 2) percentage of explants forming roots; 3) percentage of explants forming shoots; and 4) the color of the explants in each treatment after 30 days. Observational data were analyzed descriptively. The results showed that the hypocotyl response of the Barari cultivar planted in treatments P1, P2, and P3 showed cell enlargement and elongation, in contrast to explants in treatment P0 which did not show any growth response. In treatments P1 and P2, cell elongation occurred until the 10th day followed by root formation on the 15th day after planting. The percentage of explants that formed roots at P1 and P2 were 21.67% and 16.67%, respectively. In the P3 treatment, the explants only experienced cell enlargement and elongation without the formation of shoots or roots. The color of the explants on P0 treatment was slightly brown green with black tips after 30 DAP. Explants planted at P1, P2, and P3 formed shoots or shoots+roots, the hypocotyl area remained green, shoots were greenish-white, and roots were white.

Optimizing in vitro factors for improvement of shoot proliferation of ‘Flordaguard’ peach rootstock

‘Flordaguard’ has shown great potential as a standard rootstock for peach. The aim of this study was to examine the influence of Murashige & Skoog (MS) medium and modified MS-½N medium (half Nitrogen content), explant type and angle orientation on the culture medium, as well as 6-benzylaminopurine (BAP) concentrations on in vitro shoot proliferation in ‘Flordaguard’ rootstock. Explants cultured in MS-½N medium showed higher averages for number of lateral shoots and elongated shoots. The maximum average length of lateral shoots was obtained using doublephase medium. The 3.0 mg L-1 BAP induced the highest number of lateral shoots per explant (3.38 ±0.57). Whereas the higher number of lateral shoots elongated (1.75 ±0.10 cm) occurred at concentrations until 2.0 mg L-1 BAP. The study corroborated the importance of a 50% reduction in both NH4NO3 and KNO3 content in the cultured medium to improvein vitro propagation of ‘Flordaguard’ peach rootstock.

An Optimized Protocol for In Vitro Indirect Shoot Organogenesis of Impala Bronzovaya and Zanzibar Green Ricinus communis L. Varieties

The castor bean is an important industrial and ornamental crop. In the industry, it is used as a source of castor oil. Moreover, it has a large potential as a feed crop, because the seeds contain a high amount of protein. A main problem with castor bean use is the presence of toxins in the plants. Today, detoxification is carried out using various approaches, including biotechnological methods such as CRISPR/Cas9 technology. A successful application of these methods requires the availability of an efficient in vitro protocol for callus induction and shoot organogenesis. We present the results of in vitro condition optimization for two castor bean varieties (Impala Bronzovaya and Zanzibar Green). Eight different Murashige–Skoog (MS) culture media characterized by different plant growth regulator (PGR) combinations, as well as explant types (hypocotyls, cotyledonous leaves, and cotyledon petioles), were tested. The highest frequency of shoot organogenesis and average number per explant were observed during the cultivation of cotyledon petioles in both varieties on the Murashige and Skoog culture medium (MS) containing 1 or 2 mg/L of zeatin in combination with 0.1 mg/L of 3-indoleacetic acid (IAA). An optimized protocol for in vitro callus induction and shoot organogenesis may be used for biotechnological applications to obtain toxin-free castor bean, as well as Ricinus communis L. plants, with new ornamental traits and their combinations.

In vitro growth of Sempur (Dillenia philippinensis Rolfe) shoots in response to different types of plants growth regulators supplemented on MS medium

Plant tissue culture of Sempur (Dillenia philippinensis Rolfe) has not been widely reported. The previous study showed that BAP added to Murashige & Skoog (MS) basal medium promoted the growth of D. philippinensis shoot culture. This research aimed to investigate the growth of D. philippinensis shoot tip cultures on MS medium containing three types of cytokinins namely BAP, Kinetin, and 2-iP compared with Gibberelic acid (GA3). The results showed that the type and concentrations of cytokinins significantly affected all growth parameters observed except the number of roots which was only influenced by cytokinin concentrations alone. However, different levels of GA3 did not significantly affect all growth parameters observed. After eight weeks of culture, MS medium containing 1 mg/l 2-iP gave the best response for plant height, (3.68± 0.26 cm). The highest number of leaves (20.33±3.14) and nodes (6.67± 0.21) were found in MS medium containing 0.5 mg/l BAP. While the highest number of roots (1.83±0.17) was found in MS medium without the addition of cytokinins. The range of average values of plant height at different levels of GA3 was 3.08-3.74 cm; the number of leaves was 5.1-7.5; the number of nodes was 3.9-5.4 and the number of roots was 1.0-2.8.