Explant Type Research Articles

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.

The Establishment of Two Efficient Transformation Systems to Manipulate and Analyze Gene Functions in Quinoa (Chenopodium quinoa Willd.)

Quinoa (C. quinoa) is considered a gluten-free food with abundant nutrients and high tolerance to multiple abiotic stresses, which is the potential to become a major crop in future. Genetic manipulation will provide powerful tools to investigate the function and mechanism of those important genes in the regulation of quinoa development and stress responses, and further improve the quinoa in the field. However, the efficient plant transformation system for quinoa has not been well developed yet. Here, we established two rapid and efficient transformation systems for quinoa by using hairy roots and agroinfiltration of leaves, which provide useful tools for quick analysis of gene function. Hairy roots were obtained from three types of explants: cotyledon-nod with hypocotyl, cotyledon itself, and hypocotyl pieces. Interestingly, explants of cotyledon-nod with hypocotyl showed the highest transformation efficiency at 67.9%, and cotyledon displayed medium efficiency at 42.2%, while hypocotyl explants with the lowest at 31.6%. We also obtained transgenic quinoa roots successfully in-vivo, which showed low efficiency but provides a potential method to test gene functions in live plants. By using young leaves for agroinfiltration, direct injection showed a better transgenic effect compared with vacuum penetration. In juxtaposition, the transformation systems using both hairy root and leaf infiltration establish an efficient and convenient way to manipulate and analyze gene functions in quinoa, and a potential strategy for transgenic quinoa.

Establishment of Agrobacterium rhizogenes-mediated RNAi of Phryma leptostachya accelerates the functional identification of key genes of the furofuran lignan biosynthetic pathway

Phryma leptostachya is a well-known medicinal plant that produces furofuran lignans with various biological activities. However, the biosynthetic pathways of these natural products in P. leptostachya remain unclear. Hairy root cultures provide a novel method for elucidating the biosynthetic pathways of secondary metabolites. Here, we established a hairy root system for P. leptostachya by optimizing five parameters: Agrobacterium strains, explant types, infection time, A. rhizogenes cell density, and acetosyringone (AS) concentration. The highest transformation frequency (74.51 %) was achieved when the stem explants were infected with ATCC15834 (OD600 = 0.8) strain of A. rhizogenes for 30 min on 1/2 MS medium supplemented with 200 µM AS. Based on the optimal protocol, RNAi hairy roots were induced by silencing the cytochrome P450 gene, PlCYP81Q38, which converts (+)-pinoresinol to (+)-sesamin by means of (+)-piperitol in P. leptostachya, to determine its in vivo role in the lignan biosynthetic pathway and verify the availability of this system. PlCYP81Q38 silencing resulted in significant down-regulation of the PlCYP81Q38 gene at the transcriptional level, consistent with a remarkable decrease in the content of leptostachyol acetate and 6-desmethoxy-leptostachyol acetate, demonstrating its significance in the production of lignans. In conclusion, the approaches and results presented here provide a solid foundation for elucidating the role of key genes involved in the lignan biosynthesis pathway in P. leptostachya and for the industrial production of lignan through plant biotechnology.

The Application of Plant Growth Regulators in Propagation of Dendrobium Orchid with Thin Cell Layer (TCL) Technique: A Review

Plant growth regulators is one of the success factors for in vitro propagation. Plant growth regulators are organic compounds that are not nutrients and, in small amounts, promote, inhibit, or regulate physiological processes in plants. The function of growth regulators is to stimulate the growth of morphogenesis in cell, tissue and organ cultures. The most used plant growth regulators in tissue culture are auxins, cytokinins and gibberellins. One of technique for tissue culture is the thin cell layer technique. Thin Cell Layer (TCL) is a technique using small thin sections produced from organ pieces. TCL explants more effective than large conventional explants. Thin explants facilitate contact and diffusion of media into the tissue, better than thick explant slices. This literature review aims to gather information related to the application of plant growth regulators in propagation of Dendrobium orchid with thin cell layer (tcl) technique. A systematic Literature Review (SLR) uses in this paper. The growth regulators commonly used in Dendrobium orchid propagation using the TCL technique are auxins and cytokinins and the type of media that is often used in Dendrobium orchid propagation is Murashige and Skoog (MS). Protocorm Like Bodies (PLB) is the most potential explant in Dendrobium orchid propagation. The TCL technique both transverse and longitudinal has its own specificity with each type of explant used. TCL explants proved to be more effective than large conventional explants.

Development of efficient and sustainable droplet-vitrification cryoconservation protocol for shoot tips for long-term conservation of Dahlia germplasm

Dahlias are the one amongst the tuberous herbaceous plants, valued for their attractive colorful flowers with varied size cultivated due to its ornamental value as a potted plant or cut flower in many countries. Dahlia germplasm was being conserved in the in vitro Genebank of ICAR- NBPGR, New Delhi since 2005–06 under normal growth and slow growth with a regular subculturing in every 6 months and 1 year respectively, which is laborious and time-consuming. Hence, in the current study we attempted to develop an efficient droplet-vitrification cryopreservation protocol for safe and long-term conservation of dahlia germplasm. Significant effect of type and position of explants, plant vitrification solution-2 (PVS2) dehydration duration, pregrowth period and loading solution treatment duration on post-thaw regrowth after cryoconservation was observed. Shoot tips, each being 1.0 mm in length and 0.5 mm in width positioned at the tip of in vitro shoots with a similar developmental stage (similar shoot length) were excised from in vitro cultures pregrown for 6 wks on B5 basal medium supplemented with 0.5 mg/l BAP, 30 mg/l silver nitrate and 3% sucrose. The excised shoot tips were precultured on MS + 0.3 M sucrose for overnight followed by loading solution (2.0 M glycerol and 0.4 M sucrose in liquid MS medium) treatment for 80 min, PVS2 dehydration for 30 min and then transferred onto PVS2 droplets on aluminum foil strips and cryostored in liquid nitrogen (LN). An average of 44.44% post-thaw regrowth was obtained in six accessions tested and no significant difference among the accessions was observed. There was no significant loss in regrowth of cryostored shoot tips after 1 year and 6 years of cryobanking. This justifies the genotype-wide and sustainability of the protocol for cryoconservation of Dahlia germplasm using droplet-vitrification cryopreservation procedure in the cryobanks. To the best of our knowledge, this is the first report on cryoconservation of Dahlia germplasm.

Effect of Explants Type and Plant Growth Regulators on the Initiation and Multiplication of Vegetative Shoots of Stevia rebaudiana Bertoni in vitro

This study was carried out in the Plant Tissue Culture Laboratory of the College of Agriculture - Karbala Unaiversity for the period from 2022 to 2023. The study included the use of explants and different combinations in the sterilization, initiation and multiplication of the vegetative shoots of the Stevia plantiv vitro, the study was carried out in three phases: the first included sterilization of explants (shoot tips and lateral buds) using sodium hypochlorite at concentrations of (0, 0.5, 1, 1.5, 2)% for periods of (5, 10, 15) minutes. The second included the initiation of shoots cultures by cultivating the shoot tips and lateral buds on the MS medium with different concentrations (0, 0.5, 1, 1.5) mg l-1 BA and (0, 0.1, 0.2, 0.3) mg l -1 NAA, the third stage was carried out by cultivating the growths resulting from the shoot tips cultivated in the previous stage on the MS medium prepared with different concentrations (0, 1, 2, 3, 4) mg l-1 BA and (0, 0.1, 0.2, 0.3, 0.4) mg l-1NAA. The results of the study showed the superiority of the developing apical in achieving the lowest contamination rate of 38% compared to the side buds that achieved a contamination rate of 40.67%, and the concentration of 2% of NaOCl achieved the lowest pollution rate of 8.33%, and the duration of 15 minutes achieved the lowest pollution rate of 24%. The results of the initiation experiment showed the superiority of the shoot apical as a explants that achieved the highest response rate to emergence of 70% compared to the lateral shoots that recorded a response rate of 54.69%, and the concentration of 1 mg l-1 BA was significantly superior and achieved a response rate of 82.5% compared to the comparison treatment that gave a rate of 82.5%. A response of 32.5%. As for the results of the vegetative multiplication experiment, the results showed the superiority of BA at a concentration of 3 mg l-1 in achieving the highest rate in the number and length of branches was 33.35 branches Plant-1, 8.20 cm respectively. compared to the comparison treatment that achieved the lowest rate, and the concentration of 0.3 mg l-1 NAA was superior in achieving the highest average of the number and length of the vegetative total reached 30.84 plant branch-1, 7.65 cm respectively, while the comparison treatment achieved the lowest mean for the studied traits.

Effect of Explant Type and Phytohormone Concentration on Micropropagation of Citrus

Citrus fruits are among the most popular fruits in the world, thanks to their energizing flavor and health advantages.In this work, the ideal levels of activated charcoal and plant growth regulators were determined for micropropagation of lemon. Citrus limon (Osbeck Family: Rutaceae) has been successfully regenerated in vitro using both direct and indirect organogenesis in the current study. For indirect organogenesis, callus was stimulated and multiplied from leaf explants of in vitro grown seedlings on Murashige and Skoog (MS) medium with 6-Benzylaminopurine (BAP) alone or in conjunction with -naphthalene acetic acid (NAA). The placement of explants on MS medium containing BAP alone or in combination with NAA was done for direct organogenesis. For root multiplication, various concentrations of NAA were applied to well-developed microshoots. The frequency of callus induction with BAP(4µl) + NAA(0.2µl) was observed to be 94.5%. When the plants started to root, MS medium treated with 2.0 µl of IBA and 30 g/l of sucrose showed abundant root development. In order to quickly multiply disease-free potential material and take over wide regions in a short amount of time, it may be necessary to establish mature micropropagation methods for new elite lemon cultivars. This phenomena is therefore of fundamental economic relevance. In this study, it was shown that Microshoots were best rooted (90.8%) when IBA (2.0 µl) was treated for 48 hours with activated charcoal.

Effect of silver nanoparticles on tropane alkaloid production of transgenic hairy root cultures of Hyoscyamus muticus L. and their antimicrobial activity

The utilization of nanotechnology and biotechnology for enhancing the synthesis of plant bioactive chemicals is becoming increasingly common. The hairy root culture technique can be used to increase secondary metabolites such as tropane alkaloids. Agrobacterium was used to induce hairy roots from various explants of Hyoscyamus muticus. The effect of nano-silver particles (AgNPs) at concentrations of 0, 25, 50, 100, and 200 mg/L on tropane alkaloids synthesis, particularly hyoscyamine and scopolamine, was studied in transgenic hairy root cultures. Different types of explants obtained from 10-day-old seedlings of H. muticus were inoculated with two strains of Agrobacterium rhizogenes (15,834 and A4). The antimicrobial activity of an ethanolic extract of AgNPs-induced hairy root cultures of H. muticus was tested. The frequency of hairy roots was higher in hypocotyl, root, leaf, and stem explants treated with A. rhizogenes strain A4 compared to those treated with strain 15,834. In transgenic hairy root cultures, AgNPs application at a concentration of 100 mg/L resulted in the highest total tropane alkaloid production, which exhibited broad-spectrum antimicrobial activity. The study demonstrated the potential of nano-silver as an elicitor for promoting the production of target alkaloids in Hyoscyamus muticus hairy root cultures, which exhibit high biological activity.

Establishment of an Efficient Somatic Embryogenesis Protocol for Giant Reed (Arundo donax L.) and Multiplication of Obtained Shoots via Semi-Solid or Liquid Culture

This study developed an efficient protocol for the in vitro propagation of giant reed (Arundo donax L.) biomass, defining a complete cycle of the induction of somatic embryogenesis from immature inflorescences, followed by the maturation of somatic embryos and the subsequent multiplication of the derived shoots in liquid culture in a temporary immersion system (TIS). The best explants were found to be 30 cm long immature inflorescences, preferably collected in spring. Such an explant type was easy to decontaminate, and the spikelets isolated from it provided over 100 embryogenic callus lines. Among the callus induction media tested, gelled MS medium supplemented with 1.1 mg/L 2,4-D provided the highest percentage of responsive spikelets and the highest density of embryogenic callus. Maturation of the embryogenic callus was easily triggered on gelled MS medium devoid of plant growth regulators. The obtained shoots could be further multiplied on previously optimized gelled DKW medium supplemented with 30 g/L sucrose, 5 mg/L BA, 0.1 mg/L IBA, and 6.8 g/L plant agar. Subsequent high multiplication of the developed shoots was achieved in liquid culture in TIS using a Plantform™ bioreactor, with an immersion cycle of 12 min every 8 h.

In vitro regeneration of hybrid Dendrobium sect. Spatulata through pseudobulb segment culture

The low rate of Dendrobium regeneration through seed culture is a significant limitation of mass propagation development in new hybrids. An efficient protocol of in vitro regeneration through pseudobulb segment culture has been established for Dendrobium ‘Danny Dame’. Leaves and roots of seven-month-old seedlings were detached from the pseudobulb. Unsegmented pseudobulb and segments of apical, medial, and basal excised from seedlings. The four types of explants were cultured on Murashige and Skoog (MS) basal medium supplemented with different combinations of 1-naphthaleneacetic acid (NAA) and 6-benzyl amino purine (BAP). The highest number of shoots was observed in 1 mg L-1 BAP and 0.1 mg L-1 NAA using unsegmented pseudobulb explant after 2 months of culture. The unsegmented pseudobulb had a higher survivability rate and the number of shoots per explant than segmented pseudobulbs. However, if accumulated, segmented pseudobulbs can produce the number of shoots 2-4 times more than unsegmented pseudobulbs. The segment that had the highest average number of shoots was the basal segment. Two types of shoots were observed, both single shoot and multi shoot. The new shoots formation was developed from protuberance and or axillary bud. Roots were kept to grow only on the basal part of unsegmented explants and basal segment.

Research Progress and a Prospect Analysis of Asexual Bamboo Reproduction

Bamboo possesses various characteristics that make it a promising renewable biomass resource. These include rapid growth, early timber production, strong adaptability, high yield, ease of planting, wide distribution, and ease of processing. With the increasing demand for bamboo resources, rapid propagation, species selection, and breeding have always been the focus of bamboo research. However, the long and unpredictable flowering cycle of bamboo, coupled with the difficulties of obtaining seeds, has made it difficult for mass-scale propagation and the introduction of desirable traits through classical breeding methods, which hinders the genetic improvement of bamboo. Asexual propagation, tissue culture, and genetic transformation present an effective breeding method to hasten the breeding process, improve breeding efficiency, and screen and create superior new varieties, and may significantly enhance the genetic improvement of bamboo and the development of the bamboo industry. This study reviews recent research on the asexual propagation of bamboo, propagation methods, cultivation means, influencing factors, and transgenes. The bamboo species that participated in asexual reproduction were systematically sorted according to the type of explants, the formula of the culture medium, and the results achieved. In addition, the bottlenecks and development trends in each training process were identified. This study provides a reference for the rapid propagation and genetic breeding of bamboo plants.

Induction of organogenesis and callogenesis in Limbarda crithmoïdes L. (Asteraceae) explants cultured on MS media supplemented with various concentrations of Na+ and K+

The present work describes an efficient micropropagation and callus induction protocol of Limbarda crithmoïdes L. (Asteraceae), a halophyte species with medicinal and horticultural interests. The objective was to identify the culture media that support the best organogenesis and callogenic expressions of four types of aerial explants; nodal and internodal segments and leaf portions, by varying the mineral composition of twenty culture media by adding increasing concentrations of Na+ (from 0-100 mM) and K+ (from 0-50 mM). After two months of culture, parameters relating to the various expressions of organogenesis and callogenesis were measured. Results showed that the K+ and Na+ interactions affect the explants development. The combination of high concentrations of Na+ (50, 100 mM) and K+ (50 mM) ions allowed the most important regeneration of the axillary shoots (70.6-100%), root neoformation (82.6-96.1%) and callus induction (76.3-100%). Rooted plantlets with well-developed axillary shoots have been successfully acclimatized with a 93% of survival rate. The selected media would allow a large-scale multiplication of this medicinal species, without adding exogenous phytohormones, and could be used for the micropropagation of other threatened halophyte species or for the production of callus; rich in secondary metabolites.

Indirect auxiliary organogenesis of Fraxinus excelsior L. as a tool for ash dieback control

The existence of European ash (Fraxinus excelsior L.) is threatened by fungus-induced ash dieback. It is essential to find effective methods to multiply ash genotypes resistant to ash dieback while preserving the genetic diversity of these tree populations. In this paper the efficient method for production of European ash seedlings using indirect auxiliary organogenesis with multi-factor analysis of its effectiveness is presented. Procedures for a dormancy breaking treatment of seeds and effective disinfection of F. excelsior primary explants, as well as appropriate composition of the culture media taking into account impact of growth regulators and physiological gradient on the micropropagation efficiency were developed. As primary explant for micropropagation of F. excelsior, leaf buds, megagametophytes and zygotic embryos were tested. The best-performing type of primary explant for micropropagation of European ash proved to be zygotic embryos, which were successfully used to regenerate seedlings via indirect auxiliary organogenesis. No statistically significant impact of population origin of F. excelsior explant donor trees was observed on the effectiveness of callus initiation. However, such difference was significant in regard to average productivity of acquired callus cultures (number of seedlings produced) and to average root length of regenerated seedlings. Health condition of explant donor trees and their seeds affects the callus initiation rate from zygotic embryos, but does not affect the productivity of callus lines derived from the seeds and the quality of regenerated seedlings. Indirect auxiliary organogenesis of F. excelsior, developed in our study, not only provides the acquisition of ash seedlings of different genotypes, but also enables rapid selection of desired genotypes already at the callus stage. In this way, the presented method benefits not only profit oriented forestry and wood industry, but also provide the effective and fully controllable tool for reintroduction of various resistant to ash-dieback F. excelsior genotypes without loss of variability and genetic identity of its populations.

In vitro induction of callusogenesis in soryz

Purpose. Select the composition of the nutrient medium and explants for in vitro inducing the callogenesis in soryz.
 Methods. The objects were 10 varieties of soryz: ‘Helios’, ‘Saliut’, ‘Tytan’, ‘Merkurii’, ‘Odeskyi 302’, ‘Evropa’, ‘Kvarts’, ‘Samaran 6’, ‘Oktan’, ‘Fakel’. For the induction of callusogenesis, explants grown in vitro culture were selected, specifically leaves of shoots and apical parts of the root. Solid nutrient medium with agar-agar (7–8 g/l) was modified according to Murasige-Skoog (MS), Schenk-Hildebrandt (SH) and Hamburg-Eveleg (B5). To initiate callusogenesis, the nutrient medium was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6‑benzylaminopurine (BAP) in concentrations from 0.2 to 2.0 mg/l.
 Results. For all types of nutrient media, concentrations of 2,4-D of 0.4 and 0.6% were the optimal for the induction of callusogenesis. At these concentrations, 17% and 20% of callus from leaf blades and 24% and 30% from the apical parts of the root were obtained in MS nutrient medium. Lower indicators were noted for the use of SH nutrient medium: 15% and 18% in leaves and 20% and 27% in roots. The lowest percentage of induction on medium B5 was 13% and 16% and 18% and 25% by types of explants, respectively. An increase in the concentration of 2,4-D to 0.8 mg/l led to a decrease in the induction of callus mass compared to the addition of 0.4 mg/l: leaf plates – from 12% to 15%, roots – from 15% to 22%. In particular, at the studied concentrations (1.0–1.6 mg/l) on MS medium, callus was obtained in 9–12% of leaf explants and 11–17% of root explants. Analyzing the data, it is advisable to note the advantage of the nutrient medium MS, with the use of which varieties obtained from 18 to 28% of callusogenesis frequency from leaf blades. The lowest percentage of induction was found in the case of using the B5 medium: ‘Samaran 6’ – 21%, ‘Evropa’ – 20%, ‘Quartz’ – 18%, ‘Tytan’ – 17 and ‘Merkurii’ – 16%. The lowest percentage was in the varieties ‘Odeskyi 302’ and ‘Helios’ – 14%, ‘Oktan’ – 13% and ‘Saliut’ – 11%.
 Conclusions. Regardless of the concentration of 2,4-D in the nutrient medium on a hormone-free basis, induction of callusogenesis occurred more intensively in roots compared to leaves. At a concentration of 0.2 mg/l, callus formation was from 5 to 10% in leaves, and from 10 to 14% in the apical parts of the root in all studied nutrient media. Among the studied varieties, a significant advantage was noted in the variety ‘Fakel’, in which callus induction was at the level of 22–37% in different media. In MS medium, the highest rates of callusogenesis were obtained in the varieties ‘Samaran 6’, ‘Evropa’ and ‘Tytan’ (31–35%), and the lowest by ‘Saliut’, ‘Kvarts’ and ‘Helios’ (21–24%).

Tissue culture and rapid propagation technology for Gentiana rhodantha.

Gentiana rhodantha is a perennial herb of the genus Gentiana (Tourn.) L. This study was novel in establishing a regeneration system of G. rhodantha using young leaves as explants on the Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs). The roots, stems, and leaves of G. rhodantha were used as explants. The effects of the optimal explant disinfection method, type of explant used, concentrations of PGRs added to the culture media on tissue culture, and rapid propagation of G. rhodantha were studied. The results showed that the optimal disinfection method for stems and roots consisted of disinfection using 75% ethanol for 50 s, followed by 4% sodium hypochlorite (NaClO) for 10 min. The optimal disinfection technique for leaves consisted of disinfection using 75% ethanol for 50 s, followed by 4% NaClO for 8 min. Root explant was the most suitable for inducing the callus of G. rhodantha on the MS medium supplemented with different PGRs. The optimal conditions for callus induction included 1.0 mg/L 6-benzylaminopurine (6-BA) and 0.5 mg/L α-naphthalene acetic acid (NAA). The callus induction rate using the root explant reached 94.28%. MS supplemented with 2.0 mg/L 6-BA and 0.1 mg/L NAA was the optimal medium for inducing adventitious shoots from the callus of G. rhodantha. The best medium for propagation and plantlets strengthening was MS supplemented with 0.8 mg/L 6-BA and 0.3 mg/L NAA, and the propagation index was 8.62. MS supplemented with 0.3 mg/L 3-indolebutyric acid was the best culture medium for inducing the rooting of adventitious buds, with the maximum rooting rate reaching up to 100%.

Impact of different Agrobacterium rhizogenes strains on secondary metabolites accumulation in Nitraria schoberi L. hairy roots and antiviral activity of their extracts against influenza virus of subtypes A (H5N1) and A (H3N2).

To optimize protocol for obtaining hairy roots of Nitraria schoberi L. with high antiviral activities, factors such as four strain types of Agrobacterium rhizogenes (A4, ATCC15834, R-1601, 8196), two explant types, namely cotyledonous and primary leaves of seedlings, and different cultivation durations (30 and 90 d) were studied. The formation of hairy roots was observed after 2 to 4 wk of incubation, depending on the type of explant and the strain of A. rhizogenes used. The maximum transformation frequency (85.7%) was observed in the cotyledons genetically modified with the strain ATCC15834. The transgenic nature of hairy roots was revealed by PCR with primers to the Agrobacterium oncogenes rolB and rolC. The absence of contamination of the culture by A. rhizogenes was confirmed by primers to the virC and virD1 genes. Phytochemical analysis showed that accumulation of individual metabolites in the line samples exceeded their levels in the native Nitraria roots. Catechin content in the cultures of long-term cultivation (90 d) was found 1.4 to 2.2 times higher than the same samples of short cultivation (30 d) and 4.8 to 10.8 times higher in comparison with the native roots. The most productive in terms of catechin level were hairy roots of long-term cultivation obtained during the transformation of primary leaves of N. schoberi seedlings with ATCC15834 strain. These data were consistent with the highest antiviral activities against influenza viruses of A (H5N1) and A (H3N2) subtypes with neutralization indexes 6.5 to 6.75 log10, and selectivity index values were in the range 15.4 to 16.4.

Survival and State of Garlic Explants of Two Lithuanian Cultivars after Cryopreservation

Cryopreservation features of Lithuanian garlic (Allium sativum L.) cultivars ‘Žiemiai’ and ‘Dangiai’ were investigated. The survival rate and state of explants, depending on the type of explant, and dehydration conditions were evaluated in the experiment. Explants isolated from cloves and bulbils were dehydrated with a plant vitrification solution 3 (PVS 3) containing 50% w/v glycerol and 50% w/v sucrose in liquid MS medium. Three different exposure durations of 1.5, 2, and 3 h in PVS3 solution were applied at 0 °C. Unloaded stem domes were planted in Petri dishes with MS medium supplemented with 1 mg L−1 naphthaleneacetic acid (NAA) and 6–dimethylallylamino purine (2-iP) on 0.8% plant agar. The obtained results showed that the cryopreservation in liquid nitrogen reduced the survival rate of explants by 20–40%. The average number of surviving explants after freezing reached 64.3% for both cultivars. Cultivar ‘Žiemiai’ showed a 24% higher capability for survival than ‘Dangiai’. The total number of surviving explants of ‘Žiemiai’ reached 76.1%. Explants from bulbils were 23% more effective for cryopreservation compared to cloves. Evaluation of the effect of dehydration duration showed that the survival rate of the explants from bulbils of both cultivars was highest after treatment with PVS3 for 3 h and reached 91%. Treatment in PVS3 solution for 2 h was sufficient for survival of explants from cloves. Obtained results confirm that the efficiency of garlic cryopreservation depends on complex factors.

Adult Trees Cryptomeria japonica (Thunb. ex L.f.) D. Don Micropropagation: Factors Involved in the Success of the Process

Cryptomeria japonica (Thunb. ex L.f.) D. Don is a commercial tree native to Japan and is one of the most important forest species in that country and the Azores (Portugal). Because of the quality of C. japonica timber, several genetic improvement programs have been performed. Recently, some studies focusing on C. japonica somatic embryogenesis have been carried out. However, in this species, this process uses immature seeds as initial explants, and for this reason, it is not possible to achieve the maximum genetic gain (100% genetic of the donor plant). Although some studies have been made applying organogenesis to this species, the success of the process in adult trees is low. For this reason, our main goal was to optimize the micropropagation method by using trees older than 30 years as a source of plant material. In this sense, in a first experiment, we studied the effect of different types of initial explants and three basal culture media on shoot induction; then, two sucrose concentrations and two light treatments (LEDs versus fluorescent lights) were tested for the improvement of rooting. In a second experiment, the effects of different plant growth regulators (6-benzylaminopurine, meta-topolin, and thidiazuron) on shoot induction and the subsequent phases of the organogenesis process were analyzed. The cultures produced the highest number of shoots when QL medium (Quoirin and Lepoivre, 1977) and long basal explants (>1.5 cm) were used; the shoots obtained produced a higher number of roots when they were grown under red LED lights. Moreover, root induction was significantly higher in shoots previously induced with meta-topolin.

Establishment of a suitable regeneration protocol for rapid propagation of Piper nigrum L. through in vitro culture

An experiment was performed to establish a suitable regeneration protocol for the rapid propagation of Piper nigrum L. using nodal segments and leaf tissues as explants through direct and indirect organogenesis. After surface sterilization, several types of explant were inoculated onto gelled MS medium containing various concentrations and combinations of growth regulators for callus and direct shoot induction. The highest callus induction frequency was 92% and 84% in the case of leaf tissues and nodal segments, respectively, in gelled MS medium containing 2.0 mg/l 2,4-D. Multiple shoots (6.43±0.35 shoots per unit callus) were obtained when the calli from both explant types were cultured on MS medium containing 1.0 mg/l BAP. Nodal segments showed the best result (85%) in terms of direct shoot induction in MS medium supplemented with 1.5 mg/l BAP, where the highest number of shoots per explant was 5.03±0.69. The directly induced shoots were multiplied and elongated on MS medium containing 1.0 mg/l BAP and 0.5 mg/l IBA, the number and length of regenerated shoots per culture being 6.07±0.39 and 5.84±0.65 cm, respectively. The best response to root induction (86.67%) was observed when shoots were transferred to ½ strength of gelled MS medium supplemented with 1.5 mg/l IBA within 16-24 days, with 13.60±1.76 roots per shoot unit. The well-rooted shoots were successfully acclimated in a mixture of soil, sand, and compost (1:1:1) with a survival rate of 88%.
 Jahangirnagar University J. Biol. Sci. 11(1 & 2): 31-40, 2022 (June & December)

Effect of Explants Type and Plant Growth Regulators on the Stimulation and Multiplication of Vegetative Branches of Trigonella foenum-graecum L. in Vitro

The study included the use of plant tissue culture technology in the use of plant parts and different combinations in the emergence and multiplication of farms of the vegetative branches of the fenugreek plant(1,2,3)mg.l-1 BA, and the second stage was carried out by cultivating the growths resulting from the nodes grown in the previous stage on the MS medium, which was prepared with different concentrations (0, 1, 2, 3) mg.l-1 BA and (0.0,0.1,0.2,0.4)mg.L-1 NAA. The study’s results showed that the concentration of 2 mg.L-1 BA achieved a response rate of 80% compared to the peaks that did not respond. The results also showed that the same concentration of benzyl adenine achieved the highest rate in the number and length of branches, and the number of leaves reached 8.69.plant-1, 4.00 cm, 26.33 leaves.plant) respectively, compared with the control treatment that achieved the lowest rate, and the concentration 0.2 mg.l-1 NAA excelled in achieving the highest rate of the number of branches and leaves reached (7.49 branches. plant-1 and 21.50 leaves.plant-1) respectively. In comparison, the concentration of 0.4 mg.l-1 NAA excelled in achieving the highest average length of branches, reaching 3.92 cm.