Combinations Of Cytokinins Research Articles

Plant biotechnology for the conservation of Argania spinosa (Skeels L): optimising propagation and sustainable valorisation

ABSTRACT Argania spinosa, an endangered species from southwestern Morocco, is studied for preservation through plant biotechnology. The research aims to optimise argan tree propagation to boost production, preserve heritage, and use residual species. In vitro seedlings are transferred to a modified Murashige and Skoog medium with various phytohormones. Six combinations of auxins and cytokinins are tested for germination. Results show that a liquid medium with activated charcoal from argan shells promotes stem elongation. Meta-topoline at 0.5 mg L−1, combined with naphthalene acetic acid (NAA) at 0.2 mg L−1, enhances shoot growth, leaf formation, and callogenesis. Adding benzylaminopurine (BAP) at 0.1 mg L−1 and indole-3-butyric acid (IBA) at 0.02 mg L−1 to the medium elongates stems by 4.3 cm after 21 days. A 2000 ppm IBA concentration is key for a 90% rooting rate. These findings underscore plant biotechnology’s role in preserving and propagating argan trees, improving resource management, and supporting sustainability.

Effect of abiotic and biotic elicitors on vincristine accumulation in endosperm derived in vitro cultures in Catharanthus roseous (L.) G. Don

Catharanthus roseus (L.) G. Don (Apocynaceae) is a well-studied herb renowned for its in vitro culture as a source of the anti-cancer alkaloid, vincristine. However, despite the recognized advantages of triploid cells over diploid cells in terms of productivity, the triploid endosperm tissue of this important medicinal plant has not been utilized for in vitro culture initiation. In this investigation, zygotic embryos and endosperm tissues were cultured on Murashige and Skoog (MS) medium supplemented with various combinations of auxins and cytokinins. The medium containing 2.50 µM 6-Benzyladenine (BA) and 1.25 µM 2,4-Dichlorophenoxyacetic acid (2,4-D) proved to be the most effective for callus and cell culture formation. Ploidy analysis using ploidy analyzer confirmed that the endosperm-derived callus exhibited mixoploid, while the embryo-derived callus remained diploid. Liquid Chromatography Mass Spectrometry (LC–MS) analysis of callus and cell cultures grown on MS media with different combinations of auxins, cytokinins, elicitors, and precursors (both biotic and abiotic) revealed the accumulation of vincristine. Notably, treatment with a biotic elicitor derived from Aspergillus niger (300 mg/l) demonstrated superior efficacy in promoting the maximum accumulation of vincristine in endosperm-derived callus and cell biomass. These findings hold promise for the sustainable production of the anti-cancer alkaloid vincristine from endosperm-derived callus and cell cultures of Catharanthus roseus.

Effect of Plant Growth Regulators on Mass Propagation of Strawberry (Fragaria sp.)

Efficient in vitro regeneration protocols for strawberries are crucial for accelerating the propagation of superior cultivars and improving overall productivity through controlled manipulation of plant growth regulators. This study investigated the influence of different concentrations and combinations of plant growth regulators on in vitro regeneration process of strawberries. BARI Strawberry-1 runner tips were sterilized and placed on Murashige and Skoog medium (MS) supplemented with various combinations of cytokinins, including 6-benzylaminopurine (BAP) and kinetin (KIN), to initiate shoot growth. Notably, the combination of 1.5 mg/L BAP + 1.0 mg/L KIN led to the shortest duration for shoot initiation (9 days), resulting in the highest number of shoots per explants (6.33), longest shoot length (2.87 cm), and maximum number of leaves per shoot (4.00). Additionally, this combination also exhibited the highest rate of shoot regeneration (70.33%). Subsequently, the proliferated shoots were transferred to MS medium supplemented with various concentrations and combinations of auxins, specifically 3-indolebutyric acid (IBA) and 1-naphthaleneacetic acid (NAA), for root induction. The in vitro regenerated shoots cultured in MS medium supplemented with 1.5 mg/L IBA + 1.0 mg/L NAA showed the shortest duration for root formation (8 days), the highest number of roots per explants (4.00), and the longest root length (2.77 cm). Furthermore, this combination also resulted in the highest rate of root regeneration (40%). These findings contribute valuable insights for the mass production of healthy strawberry plantlets using in vitro culture techniques. J Bangladesh Agril Univ 22(2):178-184, 2024

Establishment of Efficient Protocols for In Vitro Regeneration of Hyptis Suaveolens (L.) Poit. Using Apical Shoots and Nodal Segments

The present work was undertaken with a view to standardize an efficient protocol for in vitro rapid propagation of Hyptis suaveolens. Shoot apices and nodal segments of H. suaveolens produced multiple shoot buds when cultured on MS medium supplemented with 1.5-3.0 mg/l BAP or Kn in the combinations with 0.5-1.0 mg/l IAA or NAA. Nodal explants exhibited highest proliferation (90%) and gave maximum number of shoots per culture (8.40 ± 0.18) on MS medium fortified with 1.5 mg/I BAP and 1.0 mg/l IAA. The shoot buds were elongated on MS medium fortified with different concentrations and combinations of auxins and cytokinins. Elongated shoot buds were further cultured in rooting media for root induction. One fourth MS medium -without PGR was - proved ·the most effective for - induction and proliferation of roots. The--rooted plantlets ·were 'established in the field through successive phases of acclimation and the survival rate was 70%. The Chittagong Univ. J B. Sci. 8(1&2): 99-105, 2013

Optimizing The Effects of Auxin and Cytokinin Combinations on Shoot and Root Induction of Dendrobium mussauense

Dendrobium mussaense, an orchid species of high ornamental value, was subjected to experimental treatments involving the addition of combinations of auxin and cytokinin, namely 2,4-D (2,4-dichlorophenoxyacetic acid) with BAP (6-benzylaminopurine) and 2,4-D with TDZ (thidiazuron). The research aim was to evaluate their effects on the emergence time of shoots and roots. The method used an experiment design with seven treatments. Among the treatments, the most effective was 2,4-D at a concentration of 1 mg/L combined with 0.75 mg/L of BAP. This treatment resulted in the shortest time for both shoot and root emergence. These findings underscore the importance of optimizing hormone combinations for efficient shoot and root induction in D. mussaense, providing valuable insights for orchid propagation and cultivation practices.

Exploring somatic embryogenesis in Ajuga multiflora Bunge: Profiling lipophilic metabolites via HPLC, GC-FID, and GCMS– analysis

The present study aims to assess the effect of different concentrations of auxins and combinations of plant growth regulators (PGRs), specifically auxin and cytokinin, on somatic embryogenesis in Ajuga multiflora. We also aim to compare the lipophilic metabolite profiles, such as carotenoids (CAs), fatty acids (FAs), phytosterols (PSs), and tocopherols (TPs), in globular somatic embryos (SEs), germinated SEs, and shoots of SEs derived plantlets. This assessment is carried out utilizing high performance liquid chromatography, gas chromatography-flame-ionization detection, and gas chromatography–mass spectrometry techniques. The results reveal that the application of 2,4-dichlorophenoxyacetic acid (2,4-D) had the most significant impact on somatic embryogenesis, inducing both SE initiation (34.4%) and the number of SEs (3.5) per explant. Among the explants used, leaf explants were found to be the most effective for SE induction (SEI). Additionally, the study explores the combined effect of 2,4-D and cytokinins (N6-benzyladenine, N6-BA, or 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, TDZ) on somatic embryogenesis, highlighting optimal conditions for SEI percentages and the number of SEs per explant. The maximum SE induction rate (100%) and the number of SEs (45.9) per leaf explant were obtained on an SEI medium with 30 µM of 2,4-D and 2.3 µM of TDZ. The maturation and conversion of SEs were also examined, emphasizing the role of GA3 in achieving the highest conversion percentage (94.0 ± 2.7 at 0.8 µM GA3). Furthermore, the study details the biochemical composition during various stages of somatic embryogenesis, including CAs, TPs, PSs, and FAs. Globular SEs displayed comparatively lower concentrations of CAs than their germinated counterparts and shoots from SE-derived plantlets, while the total PSs content remained relatively consistent across all stages. Conversely, fatty acids tended to accumulate more in globular SEs, with the exception of α-Linolenic acid. These findings provide valuable insights into the optimization of somatic embryogenesis protocols in A. multiflora, with implications for plant tissue culture, regeneration and lipophilic metabolites production.

Meta-topolin mediated improved micropropagation, phytochemical profiling, and assessment of genetic fidelity in Chlorophytum borivilianum Sant. et Fernand., an endangered medicinal herb

A highly effective method has been developed to examine the impact of different combination of meta-topolin and various cytokinins on Chlorophytum borivilianum Sant. et Fernand. using apical bud explants. The effects of benzyl adenine purine (BAP), kinetin (Kin), meta-topolin (mT), and thidiazuron (TDZ) were explored separately and in different concentrations (ranging from 0.5 to 3.0 mg L−1) on shoot proliferation, as well as the influence of auxins like Indole -3- acetic acid (IAA), α-Naphthalene acetic acid (NAA) and Indole-3-butyric acid (IBA) on rooting. When grown on MS medium supplemented with mT, highest growth rates were observed followed by BAP. Within the permutations tested, 2.0 mg L−1 of mT and 1.0 mg L−1 of BAP produced the best results with 96.3% success rate and average of 19.2 ± 1.92 shoots measuring 6.72 ± 0.49 cm in length after 8 weeks of culture. Among the various auxins used for in vitro root formation, 95.5% of multiple shoots effectively established roots on MS medium supplemented with 1.0 mg L−1 of IBA resulting in an average of 14.0 ± 1.39 root numbers and 3.36 ± 0.27 cm in length after 4 weeks of culture. Furthmore, 94% of the acclimated plants not only survived but also thrived when grown in a mixture of loam, sand and fym (in 1:2:1 ratio) with an average weight of 16 g. High-performance thin layer chromatography (HPTLC) analysis revealed the presence of diosgenin and stigmasterol in both in vitro regenerants and mother plant, although there were variations in their content. To ensure genetic fidelity among regenerants, ISSR and SCOT markers were used, but no somatic differences were detected. Therefore, the established procedure seems to be easy and consistent for large scale development of genetically identical clones with a greater content of bioactive compounds.

Effect of Different Plant Growth Regulators on In-vitro Micropropagation of Banana Cultivar Grand Naine (Musa spp.)

Banana (Musa sapientum) is one of the most important fruit crop cultivated in India. It is widely grown in more than 150 countries, producing on an average of 105 million tons of fruit per annum. Grand naine Banana variety is the most commonly cultivated banana with high yielding plantain. This variety mainly grown in the region of Uttar Pradesh state. Present study mainly focuses on the in-vitro micropropagation method which is described for banana cv. Grand naine. For this study, healthy sword suckers of three months old banana plantlets was used as explants. The explants was sterilized thoroughly and cultured on MS (Murashige and Skoog) media supplemented with the different concentration and combination of auxin and cytokinin like BAP, IAA and NAA which were used for the optimization of best plant growth regulator combination for multiple shoot generation and shoot growth. Among all the tested cytokinin 5 mg/l BAP along with 1mg/l IAA in medium concentration performed well and produced healthy shoots further at higher concentration of all tested cytokinin showed declining effect within the growth parameters. For the rooting purpose, MS media was supplemented with different levels of IBA, IAA and NAA. Results indicated that rooting was highly promoted in the medium supplemented with 0.5 mg/L IAA + 1.5 mg/L IBA.

Exploring the Production of Secondary Metabolites from a Halophyte Tetragonia tetragonoides through Callus Culture

Considering Korea’s gradual shift toward an aging society, consumer interest in compounds with physiological benefits, including antioxidant and anticancer effects, has surged. This study explored the potential of Tetragonia tetragonoides (Pall.) Kunze, commonly known as New Zealand Spinach (NZS), a halophyte with reported health benefits, including efficacy in treating gastrointestinal diseases, high blood pressure, diabetes, and obesity. This study also introduced a novel callus culture system for NZS, allowing for the rapid in vitro production of secondary metabolites. Optimal callus induction (100%) and biomass production (0.416 g) were achieved by adding 2.0 mg·L−1 6-BA (6-Benzylaminopurine) and 0.5 mg·L−1 2,4-D (2,4-Dichlorophenoxyacetic acid) among five auxin and cytokinin combinations. Two distinct callus types, TGC [TDZ (Thidiazuron)-supplemented Green Callus] and TNYC [TDZ + NAA (Naphthalene acetic acid)-supplemented Yellow Callus], were identified, each with unique characteristics. The calli showed total phenolic and flavonoid contents comparable to those of NZS leaves grown in the greenhouse. An expression analysis of six genes (CHS, CHI, F3H, F3′H, FLS, and DFR) involved in the kaempferol biosynthesis revealed an enhanced flavonoid biosynthesis-related gene expression in TGC, emphasizing its potential for compound production. GC-MS analysis identified distinct compound profiles in TGC and TNYC, with 2,3-butanediol and succinic acid being the predominant compounds among the nine and forty-four components, respectively. These calli offer a stable supply of functional compounds and present an environmentally sustainable solution. The derived callus culture system is anticipated to contribute to the development of healthy functional foods or pharmaceuticals from halophyte NZS.

Sesame (Sesamum indicum L.) Response to Soil Additives Applied in-Furrow at Planting

Aims: Studies were conducted to study the response of sesame to soil additives applied in-furrow. 
 Study Design: Randomized complete block with 4 replications.
 Place and Duration of Study: Field experiments were carried out during the 2016 through 2018 growing seasons in south-central Texas near Yoakum (29.27704o N, -97.12453o W).
 Methodology: Sesame seed was planted < 2.54 cm deep. Treatments were applied using a CO2-pressurized sprayer in 46.8 L ha-1 of water with one Teejet® orifice disc #45 nozzle per row immediately after seed drop but prior to furrow closure. Each plot consisted of two rows spaced 97 cm apart and 7.6 m long. Sprinkler irrigation was applied on a 2- to 3-wk schedule throughout the growing season as needed. S-metolachlor at 1.4 kg ha-1 was applied preemergence while clethodim at 0.11 kg ha-1 and diuron at 1.12 kg ha-1 were applied postemergence to control annual grasses and broadleaf weeds that were present. 
 Results: In 2016, treatments containing 7% N + 10% chelated Fe, gibberellic acid + 3-indolebutyric acid (0.045%) + cytokinin as Kinetin (0.09%), and pop-up fertilizer (9-30-0 + Zn) resulted in the greatest sesame emergence. In 2017, 2% N, bifenthrin + Bacillus amyloliquefaciens strain D747, and humic acids + Bacillus spp. resulted in greater emergence (90-97%) while in 2018, Azospirillum brasilense and 2% N resulted in the greater emergence (90-91%). In 2016, 2% N produced the greatest yield while in 2018 2% N and the 3-way combination of cytokinin as kinetin (0.090%) + gibberellic acid + indole-3-butyric acid (0.045%) resulted in up to a 117% increase in yield over the untreated. 
 Conclusion: The 3-way combination of gibberellic acid + 3-indolebutyric acid (0.045%) + cytokinin as kinetin (0.090%) and 2 % N proved to be the most consistent soil additives and resulted in a yield increases in the two years that the studies were harvested.

Efeito combinado de citocininas na propagação in vitro de três cultivares de morango

ABSTRACT In the in vitro propagation of strawberry plants, techniques have been applied to obtain disease-free plants with high multiplication rates. To improve the efficacy of micropropagation protocols, it is necessary to determine the effect of growth regulators, mainly cytokinins; however, there is no information regarding the combined use of trans zeatin (Zt) and other cytokinins for shoot induction. The objective of this work was to evaluate the effect of Zt combined with 6-benzylaminopurine (BAP), 6-furfurylaminopurine (KIN) or 2-isopentenyladenine (2iP) on the in vitro propagation of three strawberry cultivars. Combinations of cytokinins, Zt and BAP, Zt and KIN, and Zt and 2iP were proposed to evaluate shoot induction. For the acclimatization of the seedlings, two types of substrates were used (Pro-Mix, Pro-Mix plus humus 2:1). The use of Zt and KIN increased the average number of shoots per explant, and the greatest number of leaves and roots was obtained when Zt and 2iP or Zt and BAP were used. The Pro-Mix and Pro-Mix plus humus substrates did not significantly affect the number of leaves or roots, or the SPAD. These results indicate that the use of the combination of Zt and KIN allowed us to obtain the greatest number of shoots per explant that did not produce calli and promoted the development of the root system; likewise, the Pro-Mix or Pro-Mix plus humus substrate were adequate for acclimatization, allowing the growth and development of strawberry plants.

Direct Shoot Regeneration of Plumbago zeylanica Linn. through Tissue Culture Technology

ABSTRACT: Chitrak is scientifically known as Plumbago zeylanica Linn. belongs to Plumbaginaceae. Root shows medicinally valuable drug in pharmacological enterprise as the humanity is certainly prone to numerous contagious ailments due to ecological imbalance triggered by the current technology development. One of its remarkable phytoconstituents is plumbagin, a naphthoquinone molecule used to treat skin conditions, tumours, and tenacious chronic rheumatoid arthritis. The objective was to standardize the procedure of Plumbago zeylanica direct-organogenesis micropropagation. The solution contained various concentrations and combinations of auxin and cytokinins, and the nodal explant was used for quick micropropagation. The maximum multiple shoots (18.24 ± 0.51) per explant with shoot length 4. 2cm ± 0.20 cm was produced as the MS medium supplemented with B5 vitamins together with 2mg/l 6- Benzyleaminopurine (BAP) and 1mg/l Indole-3- acetic acid (IAA). The regenerated shoots were rooted in half strength MS medium with B5 vitamins that contained indole-3-butyric acid (IBA). The highest rooting frequency (100%) was seen in the 1.5 mg/l IBA-containing rooting media. The extreme roots were 9.2±0.14 per explant. The average root length was 4.2± 0.10 cm. The in vitro rooted Shoots were then transplanted in the field showing a 100 percentage survival rate.

Optimization of Different Auxin and Cytokinin Combination in Nutrient Medium for Establishment of Optimal in vitro Multiple Plantlet in Ficus carica L. cv Siyah Orak

Ficus carica Linnaeus is a flowering plant under the Moraceae family, usually propagated conventionally from cuttings due to the seeds being non-viable. However, this method is prone to diseases, and pests, time-consuming and space-intensive. Therefore, other methods are needed to overcome these issues. This study was conducted to induce callus and multiple shoots via plant tissue culture techniques enabling mass production of fig plants. Initially, leaf segments of Ficus carica L. cv Siyah Orak were cultured on different MS media strengths (¼, ½, ¾,1 MS) to induce callus. The highest callus means weight was observed on explant cultured in ¾ MS media (875±0.036). Callus was proliferated by subculturing explant into ¾ MS media supplemented with different concentrations of TDZ (0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/L). MS media (3/4) supplemented with 2.0 mg/L TDZ (920±0.03) shows the best result for callus proliferation. Callus induction using transverse and longitudinal thin cell layers from nodal segments cultured on different MS media strengths (¼, ½, ¾,1 MS) shows ¼ MS as the optimum media for both tTCL (100±0) and lTCL (96.7±0.15). Friable callus (%) was observed the highest on ½ MS (63.33±0.55) and ¼ MS (76.67±0.50) media for both tTCL and lTCL, respectively. As for the number of leaves produced, both tTCL (0.83±0.0.28) and lTCL (1.00±0.33) explant showed the best results in ¼ MS media. Apical buds produced the highest mean for both the number of leaves and length of the shoot on 1MS media supplemented with 2.0 mg/L BAP (3.5±0.20, 13.73±0.66), respectively. For root formation (%) and number of roots, both show the best results in media supplemented with 2.5 mg/L IAA (10±0.31, 0.83±0.50). It can be concluded that the best shoot growth performance was observed from apical bud cultured on 1MS media supplemented with 2.0 mg/L BAP+ 2.5 mg/L IAA.

Ex vitro shoot induction of rodent tuber (Typhonium flagelliforme) with benzyl amino purine and naphthalene acetic acid

A medicinal plant called rodent tuber has the potential to both prevent and treat various diseases. There is no Standard Operating Procedure (SOP) governing the growth method of rodent tuber because it still grows wild and is rarely cultivated. This research aimed to study the optimal growth of rodent tuber for mass seed propagation based on the type of explants and the concentration of auxin and cytokinin combinations for inducing shoots. While generative parameters (harvesting of tubers/shoots) were observed up to 13 WAP (Week After Planting), vegetative parameters were observed up to 10 WAP. The results showed that the best type of explants for shoot induction and growth of rodent tuber were whole plants with a survival rate of 100%; the number of shoots was 6.65; the plant height was 21.27 cm with 6.34 leaves; 26.56 roots with a length of 16.05 cm; and 40.80% flowering plants. PGR (Plant Growth Regulator) combination of 50 ppm BAP (Benzyl Amino Purine) and 100 ppm NAA (Naphthalene Acetic Acid) became the best explant soaking solution by forming eight shoots and the height of plants was 24.13 cm with seven leaves. The explant produced the highest root length in a combination of 100 ppm BAP and 50 ppm NAA, which was 17.70 cm. The interaction between whole plant explants immersed in 0 ppm BAP and 100 ppm NAA solution resulted in the most significant number of roots (41). Those goals can support the SOP of propagated methods and ensure the consistency, quality, and availability of raw materials for traditional medicine.

Growth of arabica coffee seeds (Coffea arabica L.) Sigarar Utang Variety as a response of treatment of various growth regulators and types of shade

Productivity of arabica coffee (Coffea arabica L.) Sigarar Utang Variety in Indonesia is still relatively low, especially in Simalungun Regency, Sumatera Utara. Some factors that need to be considered in nurseries are growth regulator and shade. This study aims to find out information on the type of growth regulators and the best shade density for the growth of arabica coffee seedlings. The experiment was carried out from March to June 2022 in Nagori Ujung Bawang, Simalungun Regency and Research Laboratory, Faculty of Agriculture, Universitas Sumatra Utara. The experiment used a Split Plot Design consisting of 2 factors and repeated 3 times. The first factor (main plot) consists of 4 levels, namely without shade, 50%, 70%, and 90% shade, while the second factor (sub-plot) consists of 3 levels are auxin growth regulator, cytokinin, and the combination of auxin with cytokinin. The results showed that the interaction of shade treatment and growth regulators had a significant effect on root canopy ratio. Root canopy ratio had a significant effect on the interaction of 90% shade treatment and a combination of auxin cytokinin of 5.27 g.

The Effects of Different Auxin–Cytokinin Combinations on Morphogenesis of Fritillaria meleagris Using Bulb Scale Sections In Vitro

Fritillaria meleagris is a horticulturally and medicinally valuable bulbous plant that requires a period of low temperatures for proper growth and flowering. Since conventional methods of propagation are ineffective and very slow, tissue culture techniques offer an integrated approach to mass production of this valuable geophyte. In this study, we investigated the effects of various auxin–cytokinin combinations on different morphogenetic pathways in bulb scale culture. Bulbs obtained in vitro were cut longitudinally, and bulb scales were cultured for four weeks at 7 °C on MS medium supplemented with 6-benzylaminopurine (BAP) in combination with 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA) at different concentrations in order to investigate the influence of plant growth regulators (PGRs) on different morphogenetic responses. Regeneration percentage, number of shoots per explant, shoot length, number of bulbs and number of somatic embryos were monitored weekly. After chilling, bulb scales were transferred to 24 °C, and all parameters were recorded again. Low PGR concentrations were very effective for shoot multiplication, yielding up to 5.5 shoots per explant. 2,4-D (at 2 mg/L) in combination with low BAP (0.25 mg/L) produced the highest number of bulbs (11.00 ± 0.00), while PGR-free medium was extremely effective in somatic embryo formation (13.50 ± 2.90). Detached somatic embryos and bulblets continued to grow and develop on fresh PGR-free medium. We present data demonstrating that low auxin–cytokinin concentrations and PGR-free medium provide an effective method for a combined morphogenetic pathway in F. meleagris that is suitable for large-scale propagation.

Shoot Culture of Ocimum sp. and Its Phytochemical Profile

The success of the in vitro culture technique is influenced by many factors, including the type of explants, culture medium and exogenous plant growth regulators (PGR). These factors greatly influence the speed and effectiveness of plant regeneration as well as the profile of the phytochemical compounds produced. Therefore, this study aims to determine the effect of the PGR combination on the growth response of in vitro node explants as well as the phytochemical profiles of Ocimum sp. Node explants obtained from 2 weeks old in vitro seedlings were cultured on MS medium with the addition of PGR, namely BAP and Kinetin (0.2 and 5 mg/l) combined with NAA (0.1 and 0.2 mg/l) and synthetic cytokinins alone namely Thidiazuron (TDZ) (1, 3, and 5 mg/l). The growth response of the explants and the potential for regeneration were observed for 8 weeks of culture. Effects of adding activated charcoal (AC) to root media were observed on growth of plantlets aged 2 weeks. Phytochemical profile of In vitro shoot was analyzed using GC-MS and LC-MS to be compared with its profile in in vitro callus tissue. The results showed that MS medium with the addition of a combination of cytokinin and auxin was able to induce shoot regeneration in node explants of Ocimum sp. in vitro. The Kinetin/NAA combination produced better shoot height growth, while the BAP/NAA combination produced a higher leaves number. Thidiazuron at all concentrations was able to induce shoots that were more likely to form rosettes. The addition of AC to the rooting medium did not have a positive effect on the response of shoot and plantlet growth. Chromatographic screening showed different profiles of secondary compounds in the callus and shoot tissues of Ocimum sp. in vitro. Callus composed of actively dividing cells do not produce some of the secondary compounds as produced by in vitro shoots. This shows the difference in the potential of cells or tissues in synthesizing secondary metabolites.

Conservation in Tissue Culture of Malacocarpus crithmifolius (Retz.) Fisch. C.A.Mey. - Relict Species from Mangyshlak

Malacocarpus crithmifolius is an endangered rare relict with small size populations grown in a limited area. The purpose of this study was to initiate in vitro and micro replication of Malacocarpus crithmifolius. The vegetative and reproductive explants and Murashige & Skoog medium (MS) supplemented with different concentrations and combinations of cytokinin and auxins were used. The response reaction of different explants and the plant growth regulators' effects on shooting frequency, the number of shoots, callusogenesis, and rooting percent were investigated. Culturing generative explants on the MS medium containing 6-Benzylaminopurine (BAP) combined with indole-3-acetic acid (IAA) or indole-butyric acid (IBA) led to direct shoot regeneration. The bud mass appeared in the leaf axil under inflorescences after one week of cultivation on the initiated medium. The replication was reached by micro-cutting of induced additional shoots and 3-fold passaging them on the fresh mediums. The average multiplication result at MS with BAP in combination with 1-Naphthaleneacetic acid (NAA) or IAA composed 5.85 after three months of cultivation. In conclusion, the applicable explant and phytohormonal composition of nutrient medium for initiation and micropropagation of M. crithmifolius were developed.

Efficient and rapid system of plant regeneration via protoplast cultures of Fagopyrum esculentum Moench

In the present study, a high yield of isolated protoplasts from the agronomically important crop Fagopyrum esculentum was obtained by applying a mixture of cellulase, pectolyase, and driselase. We demonstrated that the yield of morphogenic callus-derived protoplasts was 1 × 106 protoplasts per g of fresh tissue. For hypocotyls used as the protoplast source, the number of released cells was twice lower. The protoplasts, embedded in an agarose matrix and cultured in a modified Kao and Michayluk media supplemented with phytosulfokine, re-enter the cell cycle and start to develop, forming microcalli. The plating efficiency was about 20% in the case of hypocotyl- and morphogenic callus-derived protoplasts. For plant regeneration, the medium was supplemented with different combinations of cytokinin. Somatic embryogenesis and organogenesis occur during the cultivation of the protoplast-derived tissues, depending on the applied protoplast source. For the first time, an effective protoplast-to-plant system for F. esculentum has been developed.

Response of various growth regulators and shade intensity to the growth of arabica coffee seedlings of Sigarar Utang variety

Abstract. Damanik RIM, Hanum C, Sembiring LJ. 2023. Response of various growth regulators and shade intensity to the growth of arabica coffee seedlings of Sigarar Utang variety. Nusantara Bioscience 15: 85-89. The productivity of Arabica coffee (Coffea arabica L.) of the Sigarar Utang variety in Indonesia is still relatively low, especially in Simalungun District, North Sumatra Province, Indonesia. Growth regulators and shade are some factors that must be considered in nurseries. This research aims to determine the best growth regulators type and shade intensity for Arabika coffee seedling growth. The experiment was conducted from March to June 2022 in Nagori Ujung Bawang, Simalungun District, and the Central Laboratory of Agriculture Faculty, Universitas Sumatera Utara, Medan, North Sumatra. The study used a Split Plot design of 2 factors and 3 replications. The first factor (main plot) consists of 4 levels, namely 0, 50, 70, and 90% shade intensity. The second factor (sub-plot) consists of the three growth regulator treatments, i.e., auxin, cytokinin, and the combination of auxin and cytokinin. The results showed that the interaction between shade intensity and growth regulators had a significantly different effect on increasing root length. The shade treatment significantly affected plant height, leaf area, and total chlorophyll content of leaves. However, it did not affect the stem diameter. A shade intensity of 90% significantly increased plant height, leaf area, and total chlorophyll content of leaves.