Shoot Proliferation Research Articles

In vitro multiplication of peach rootstocks

The leaf yellowing and senescence during micro-propagation reduces the shoot proliferation rates with every sub-culture in peach rootstock (Prunus spp.). The effect of culture media and supplements on the proliferation of cultures during the micro-propagation of two peach rootstocks ‘Sharbati’ and ‘Flordaguard’ was studied. In both Prunus genotypes, the proliferated cultures decreased from 77.3% and 67.0 % after the first sub-culture to 35.3 % and 27.3 % after the third sub-culture in ‘Sharbati’ and ‘Flordaguard’, respectively. QL medium significantly improved the proportion of proliferated cultures over MS, WPM, DKW and the modified MS media. The highest proliferated cultures (79.0% and 70.7%) and shoot number per culture (4.2 and 3.7) were recorded after the third subculture, in ‘Sharbati’ and ‘Flordaguard’, respectively with QL medium. Both the rootstocks varied in their response to iron chelates. The higher proliferation rates were obtained with MS medium by substituting Fe-EDTA (0.1 mM) with Fe-EDDHA (0.1-0.3 mM) in ‘Sharbati’ and with Fe-Na EDTA (0.1 mM) in ‘Flordaguard’. In‘Sharbati’, the highest proliferated cultures (44.3%) after the third subculture were observed with Fe-EDDHA (0.3 mM). In ‘Flordaguard’, the highest proportion of proliferated cultures (45%) after the third subculture was recorded with Fe-Na EDTA (0.1 mM). Adding silver nitrate (3 mM) improved the shoot proliferation rates to the highest levels (63% and 51%) after third subculture in ‘Sharbati’ and ‘Flordaguard’, respectively. The highest rooting (34.3% and 38.0%) and number of roots per shoot (2.2 and 2.5) were recorded with 3.75 mM of IBA in ‘Sharbati’ and ‘Flordaguard’, respectively.

In vitro and ex vitro propagation of Turkish myrtles through conventional and plantform bioreactor systems.

The myrtle (Myrtus communis) plant naturally grows in the temperate Mediterranean and subtropical regions and is used for various purposes; thus, it is among the promising species of horticultural crops. This study aimed to evaluate and compare the performance of different propagation systems, including rooting, solid media propagation, rooting, and with the Plantform bioreactor system, in achieving healthy and rapid growth of four myrtle genotypes with diverse genetic origins and well-regional adaptation. The selection of myrtle genotypes with distinct genetic backgrounds and proven adaptability to specific regions allowed for a comprehensive assessment of the propagation systems under investigation. Present findings proved that the Plantform system, the new-generation tissue culture system, was quite successful in micropropagation and rooting myrtle genotypes. We succeeded in vitro micropropagation and rooting of diverse wild myrtle genotypes, enabling year-round propagation without reliance on specific seasons or environmental conditions. The process involved initiating cultures from explants and multiplying them through shoot proliferation in a controlled environment. This contributes to sustainable plant propagation, preserving and utilizing genetic resources for conservation and agriculture.

Conservation and Large Scale Propagation of a Valuable Ornamental Aquatic Plant, Echinodorus argentinensis Rataj through Micropropagation

A protocol was standardized for large scale propagation through multiple shoot proliferation from the basal buds of Echinodorus argentinensis, an ornamental aquatic plant. Explants were treated for 15 minutes with commercial bleach (containing 0.825% Sodium hypochlorite as active ingredient) which was diluted with distilled water to a concentration of 15%; and were then dipped for a second in 100% ethanol to obtain axenic cultures. The axenic basal buds initiated shoot formation within a week when cultured on full strength Murashige & Skoog medium (MS medium) supplemented with 1.0 mg/l 6-Benzylaminopurine (BAP). Number of shoots produced per explant was maximum (2.5672 shoots per explant p< 0.05) on MS medium supplemented with 1.0 mg/l BAP. Shoots were elongated considerably in all the media tried for shoot multiplication and maximum elongation (2.87 cm, p< 0.05) of the in vitro developed shoots was observed on MS medium supplemented with 1.0 mg/l BAP in combination with 0.1 mg/l Naphthalene acetic acid (NAA). Root regeneration was observed on all the shoot regeneration media and the highest number of roots/shoot (3.4568 roots per shoot) was obtained on MS medium devoid of any kind of plant growth regulators. The plantlets developed in vitro were subjected to acclimatization procedure and were finally planted in the aquarium. The protocol is ready for commercialization.

El rol del AIA y BAP en la regeneración y formación de brotes in vitro de tres variedades de fresa (Fragaria x ananassa Duch.)

The objective was to determine the response of three strawberrry varieties (Fragaria x ananassa Duch.) to the aplication of two plant growth regulators: indol acetic acid (IAA) and 6-Benzyl aminopurine (BAP) added to Murashige & Skoog médium in strawberry micropropagation. The complete randomized block design was used and three strawberry varieties ‘Aromas’, ‘Albion’ and ‘Camarosa’ were considered as trials. MS medium for shoot regeneration in initial stage was supplemented with BAP 0.5 mgL-1 and IAA 0.6 mgL-1. Shoot proliferation MS medium was supplemented with BAP 0.5 mgL-1 in multiplication stage and MS medium hormone free was used for rooting induction. It was determined that after 32 days of cultivation the combination of IAA and BAP significantly affected shoot regeneration of apical shoot tips of three strawberry varieties. The greatest effect was caused on ‘Camarosa’. In addition, results on shoot multiplication using BAP indicated that ‘Albion’ performed better in shoot proliferation in terms of shoots per explants reaching 2.51 compared to ‘Camarosa’ and ‘Aromas’ with significant differences. However rooting medium with no growth regulators improves root development with best response of ‘Camarosa’ root length reaching 4.76 cm.

Optimization of an Efficient Micropropagation Protocol and the Assessment of Genetic Fidelity of Solanum tuberosum L. cv Kufri Neelkanth

The aim of our experiment was to determine the optimal types and concentrations of plant growth regulators used during different stages of micropropagation and assess the genetic fidelity of tissue culture raised plants of a purple colored cv Kufri Neelkanth by using reliable markers. According to the shoot bud initiation data, it was found that utilizing a combination of 0.25 mg l-1 BAP and 0.5 mg l-1 Kinetin resulted in the greatest number of shoots per explant (3.5±0.12) within a timeframe of (3.3±0.08) days. The maximum number of in vitro shoots per shootlet (10.4±0.59) were recorded when auxins were used in combination with NAA (0.01 mg l-1 NAA) and Kinetin (0.25 mg l-1) for shoot proliferation. In vitro root initiation was observed in (2.1±0.07) days on MS medium fortified with 2.5 mg l-1 NAA. The maximum number of in vitro roots per shoots (13.0±0.55) were observed when MS media fortified with 2.5 mg l-1 IBA. Maximum 100% rooting was observed in all MS media supplemented with different concentrations of auxins. In vitro raised plants were assessed for genetic fidelity by using twenty RAPD primers (genetic markers). Out of twenty primers used only four primers produced amplification. DNA banding patterns of all tissue culture raised plants and mother plants were monomorphic showing true to type planting material. This protocol for tissue culture propagation along with testing its genetic fidelity could be useful for better conservation of germplasm and genetic transformation studies in potato.

Physio-Biochemical Integrators and Transcriptome Analysis Reveal Nano-Elicitation Associated Response during Dendrocalamus asper (Schult. and Schult. F.) Backer ex K. Heyne Micropropagation.

Bamboos are perennial, arborescent, monocarpic and industrially important non-timber plants. They are important for various purposes, such as carbon sequestration, biodiversity support, construction, and food and fiber production. However, traditional vegetative propagation is insufficient for bamboo multiplication. Moreover, little is known about the mechanism of gold nanoparticles (AuNPs) in vitro proliferation and regulation of physiological and biochemical properties. In this study, we investigated the impacts of citrate and cetyltrimethylammonium bromide (CTAB) coated AuNPs on in vitro proliferation, photosynthetic pigment content and antioxidant potential of Dendrocalamus asper (Schult. and Schult. F.) Backer ex K. Heyne. Various morpho-physiological and biochemical parameters were differentially affected along the citrate- and CTAB-coated AuNPs concentration gradients (200-600 µM). In vitro shoot proliferation, photosynthetic pigment content and antioxidant activities were higher in D. asper grown on Murashige and Skoog medium supplemented with 2 mg·L-1 benzyladenine and 400 µM citrate-coated AuNPs than in those grown on Murashige and Skoog medium supplemented with 600 µM CTAB- coated AuNPs. Identification of genes regulating in vitro D. asper proliferation will help understand the molecular regulation of AuNPs-mediated elicitation for modulating various physiological and biochemical activities during micropropagation. Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway analyses identified differentially expressed genes associated with in vitro modulation of AuNPs-regulated biological processes and molecular functions. The findings of this study provide new insight into AuNPs-mediated elicitation of in vitro mass scale bamboo propagation.

Influence of Plant Growth Regulators and Dark Pretreatment on Shoot Proliferation, Rooting and Acclimatization of Thorny Blackberry

Influence of Plant Growth Regulators and Dark Pretreatment on Shoot Proliferation, Rooting and Acclimatization of Thorny Blackberry

First Report of Candidatus Phytoplasma asteris (16SrI-B subgroup) associated with phyllody disease of fenugreek in the world.

Fenugreek (Trigonella foenum-graecum) is a leafy vegetable and spice crop, native to Indian subcontinent and Eastern Mediterranean region. Phytoplasma infection symptoms were observed in fenugreek at ICAR-National Bureau of Plant Genetic Resources Regional Station, Jodhpur and Agricultural Research Station Mandore Jodhpur, Rajasthan, India. The first appearance of phytoplasma suspected symptoms of little leaf was recorded after 50 days of sowing in the months of January 2022. The major symptoms recorded were virescence, phyllody, shoot proliferation, witches-broom, little leaf, yellowing and overall stunted growth in 146 germplasm accessions at NBPGR research farm, Jodhpur and one major commercially cultivated variety RMT 305 at Mandore Jodhpur. Ten samples from symptomatic and five samples from asymptomatic fenugreek plants were collected and processed for total DNA extraction using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) and secA gene specific primers (SecAfor1/SecArev3 and SecAfor2/SecArev3) (Schneider et al. 1995; Gundersen and Lee 1996; Hodgetts et al. 2008). The amplicons of ∼1.25 kb with 16S rRNA and ∼480 bp with secA gene specific primers were amplified in all symptomatic fenugreek samples. In negative control (asymptomatic plants) no amplification was observed with either of gene specific primers in gel electrophoresis. PCR amplified products from the six selected positive samples (FPP-NBPGR-J-01 to FPP-NBPGR-J-04 and FPP-MND-01 to FPP-MND-02) of 16S rRNA and secA gene, were sequenced from both ends. Sequences were deposited in the NCBI GenBank with accession numbers ON756108-ON756113 for 16S rRNA gene sequences and ON745809 to ON745814 for secA gene sequences. BLAST analysis of 16S rRNA and secA sequences revealed 100% sequence identity among themselves and 99.95 to 100% sequence identity with the earlier reported phytoplasma strains of aster yellows group related phytoplasma strains (GenBank Acc. No. MN239504, MN080270) belonging to Ca. P. asteris (16SrI group). Further analyses of the 16S rRNA and secA gene-based phylogenetic tree and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene study demonstrated that the phytoplasma associated with fenugreek phyllody belonged to 16Sr group I ('Ca. P. asteris') and subgroup B (GenBank accession AP006628), with similarity coefficient of 1.0. Earlier association of 16Sr-II-D subgroup (Ca. P. australasiae) with fenugreek as host was reported from Pakistan (Malik et al., 2020). To the best of our knowledge, this is the first report of a 'Ca. P. asteris', 16SrI-B subgroup related phytoplasma strain associated with fenugreek phyllody in the world. The 16SrI-B phytoplasma strain is a widely distributed strain associated with several agricultural and horticultural crops of India (Rao 2021). This is not only the first instance of fenugreek phyllody disease found in India, but also the first instance of fenugreek phyllody caused by 16SrI-B subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as fenugreek is one of the most common seed spice crop being grown all over India.

Double phase culture system mediated enhanced protocol for shoot proliferation of Vanilla andamanica Rolfe. - an endemic wild relative of commercial Vanilla from the Andaman and Nicobar Islands

A liquid overlay culture system in micropropagation improved shoot proliferation in many species. In the present investigation, we have developed an enhanced shoot proliferation protocol using the double phase culture system (DPS) for Vanilla andamanica, an endemic species of the Andaman and Nicobar Islands recognized under the vulnerable category by the IUCN. In vitro generated nodal explants were used for shoot proliferation experiments and were tested in Murashige and Skoog medium augmented with various cytokinins (BAP, KIN, 2iP) and auxins (IBA, NAA, IAA) to produce a maximum of 3.24 ± 0.08 shoots per explant in 5 µM BAP and 5.23±0.67 roots per shoot in basal MS medium. A remarkable increase in shoot regeneration was observed when the nodal explant was cultured on DPS system with optimum cytokinin (5 µM BAP). On evaluation of the influence of DPS and conventional single-phase systems (SPS), (solid medium) exhibits an improved multiplication rate on a DPS with optimal BAP (5µM) with an average of 8.66 ± 0.17 shoots per explant, which represents a 2-fold increase over the rate of SPS + 5 µM BAP. The plantlets were rooted at 100% frequency in half-strength MS medium devoid of auxins and acclimatized with 100% success. A double-phase mediated enhanced shoot proliferating procedure could be employed for large scale multiplication and commercial breeding trials for better results with regard to V. andamanica.

Improvements in morphometric and structural traits of Vitex trifolia L. to exogenous application of nano-silicon in vitro

The present study aimed to analyze the effect of silicon nanoparticles (SiNPs) on the in vitro proliferation of shoots, biomass production, foliar micro-structural features, and root-inducing ability in Vitex trifolia L. Bud breaking was achieved from axillary meristems on 2.5 mg L−1 of 6-benzylaminopurine (BAP) in Murashige and Skoog's (MS) medium. The shoots were proliferated (18.0 ± 0.39 shoots) on 0.75 mg L−1 BAP with 0.2 mg L−1 1-Naphthaleneacetic acid (NAA). Among the varied concentrations of Aerosil300 (hydrophilic silica) used, 6.0 mg L−1 SiNPs with optimal growth regulators into the medium had a positive impact on shoot proliferation (24.0 ± 0.30 shoots) and fresh biomass production. The light microscopic evaluation showed that the leaves developed without SiNPs possessed underdeveloped cuticle, trichomes, stomata, epidermal cells, and photosynthetic and vascular tissues. The SiNPs treatment resulted in well-differentiated tissue systems and favored an increase in width of the lamina, differentiation of palisade and spongy tissues, thickening of cell walls, increased density of trichomes, organized guard cells in stomata, and improved xylem and phloem tissues. The shoots were effectively rooted on half-strength MS medium containing 3.0 mg L−1 NAA, and the shoots derived from SiNPs treatment had the highest rooting efficiency (8.0 roots per shoot). The findings highlight that the use of SiNPs significantly promoted tissue systems and morphology of V. trifolia, which favored the survival of plantlets under in vivo conditions.

Jujube witches' broom phytoplasmas inhibit ZjBRC1-mediated abscisic acid metabolism to induce shoot proliferation.

Jujube witches' broom (JWB) phytoplasmas parasitize the sieve tubes of diseased phloem and cause an excessive proliferation of axillary shoots from dormant lateral buds to favour their transmission. In previous research, two JWB effectors, SJP1 and SJP2, were identified to induce lateral bud outgrowth by disrupting ZjBRC1-mediated auxin flux. However, the pathogenesis of JWB disease remains largely unknown. Here, tissue-specific transcriptional reprogramming was examined to gain insight into the genetic mechanisms acting inside jujube lateral buds under JWB phytoplasma infection. JWB phytoplasmas modulated a series of plant signalling networks involved in lateral bud development and defence, including auxin, abscisic acid (ABA), ethylene, jasmonic acid, and salicylic acid. JWB-induced bud outgrowth was accompanied by downregulation of ABA synthesis within lateral buds. ABA application rescued the bushy appearances of transgenic Arabidopsis overexpressing SJP1 and SJP2 in Col-0 and ZjBRC1 in the brc1-2 mutant. Furthermore, the expression of ZjBRC1 and ABA-related genes ZjHB40 and ZjNCED3 was negatively correlated with lateral main bud outgrowth in decapitated healthy jujube. Molecular evidence showed that ZjBRC1 interacted with ZjBRC2 via its N-terminus to activate ZjHB40 and ZjNCED3 expression and ABA accumulation in transgenic jujube calli. In addition, ZjBRC1 widely regulated differentially expressed genes related to ABA homeostasis and ABA signalling, especially by binding to and suppressing ABA receptors. Therefore, these results suggest that JWB phytoplasmas hijack the ZjBRC1-mediated ABA pathways to stimulate lateral bud outgrowth and expansion, providing a strategy to engineer plants resistant to JWB phytoplasma disease and regulate woody plant architecture to promote crop yield and quality.

Induction of polyploidy in Periwinkle (Catharanthus roseus (L.) G. Don) by in vitro treatment with colchicine

Periwinkle (Catharanthus roseus (L.) G. Don) is an important medicinal plant that can be used for producing alkaloids such as vinblastine and vincristine for treatment of cancers, especially blood cancer. This research induced polyploid C. roseus through tissue culture. Experiments were conducted to find suitabe media for in vitro shoot multiplication. Murashige and Skoog (MS) medium supplemented with 1 mg/l BAP, 0.2 mg/l NAA and 10 mg/l AdS showed a better response in terms of the emergence, proliferation and multiplication of shoots. The in vitro shoots were immersed in colchicine solution with different concentrations (0%; 0.025%; 0.05%; 0.075%; 0.1%) for 24 to 72 hours. Results showed that Colchicine concentration (0,025%) was the most effective measure within 48 hours. The ploidy level of the colchicine-treated individuals was analysed by counting the number of stomatas, counting the number of chromosomes and measuring nuclear DNA by flow cytometry.

Effect of BAP (6-Benzylaminopurine) and NAA (α-Napthalene Acetic Acid) treatment on Micropropagation of Adansonia digitata L.

The African Baobab, also known as Kalpvriksha (Adansonia digitata L.), is a significant multi-purpose tree that has gained popularity among people, primarily in African countries, for its use as food, fodder, clothing, and ropes. There is one of the obstacle arises in growing this plant by farmers in India is the lack of planting materials. The young plants of A. digitata L. have to be imported form selected nurseries. Therefore, micropropagation can be used as a substitute method to create offspring that are genetically similar to the parent plant. In order to micropropagate Kalpvriksh (A. digitata L.), this experiment is being conducted utilizing shoot tip explants. For shoot proliferation, these explants are grown in MS medium containing various concentrations of BAP (6- Benzylamino purine): 0.5, 1.0, 1.5, 2.5, 5.0 mg/L. Shoots are rooted in a medium containing at the same concentration of NAA (α-Napthalene Acetic Acid) 0.5 mg/L alone or with combination. Complete Randomized Design (CRD) is used to conduct each experiment. ANOVA and the Duncan New Multiple Range test are used to examine the data. According to the technique for micropropagation of A. digitata L. The concentration of BAP and NAA was found to be 1.5 mg/L and 0.5 mg/L respectively which showed better enhancement for shoot regeneration and rooting for micropropagation of Kalpvriksha (Adansonia digitata L.) tree.

Optimization of explants, media, plant growth regulators and carbohydrates on callus induction and plant regeneration in Citrus jambhiri Lush.

Callus induction was attempted from the four explants viz. root, cotyledon, epicotyl, and leaf segments excised from in vitro raised seedlings of C. jambhiri. Among various MS media supplementations with growth regulators and carbohydrates, the maximum (95.50%) and the earliest (8.30 days) callogenesis was obtained in epicotyl segments, when cultured on MS medium supplemented with NAA (10.0 mgl-1) + BAP (1.0 mgl-1) + sucrose (8%). The modified MS (macro and micro-nutrients reduced to half) fortified with BAP (5.0 mgl-1) + GA3 (3.0 mgl-1) recorded maximum shoot regeneration (43.10%) from callus, with an average of 5.30 shoots per callus after 35.50 days of culturing. However, prolonged exposure to GA3 resulted in thin elongated shoots and leaves. The age of the callus substantially influenced the plant regeneration frequency. The potency of the callus to regenerate decreased significantly with an increase in the age of the callus. Shoot regeneration was recorded maximum (43.43%) in 60 days old calli, followed by 90 days old (30.48%) calli, whereas it was minimum (10.46%) in 150 days old calli. The maximum (79.50%) shoot proliferation was recorded in MS medium supplemented with BAP (1.0 mgl-1) + Kin (0.5 mgl-1) with an average of 5.06 shoots per culture. The MS medium fortified with NAA (1.0 mgl-1) + IBA (1.0 mgl-1) induced maximum (77.33%) rooting, with an average of 3.19 roots per shoot after 13.4 days of culturing. Rooted plants were hardened and survived the best (83.6%) on the potting mixture consisting of cocopeat + vermiculite + perlite (2:1:1).

Optimization of Hormonal Compositions of Media in In Vitro Propagation of Orange Cultivars from Shoot Tip Nodal Segments

Orange is one of the most horticulturally important and widely cultivated Citrus species. Citrus cultivar improvement via conventional breeding strategies is normally impeded by factors related to its reproductive biology. The present study was undertaken to investigate the optimization of growth regulators’ composition of media in in vitro propagation of orange cultivar from nodal segment explants. The nodal segment explants were collected from sweet orange cultivars. The MS medium supplemented with sucrose and different concentrations of growth regulators were used for shoot proliferation and root induction. The optimum compositions of growth regulators in MS medium were assessed. The result indicated that the highest shoot response was recorded for Washington naval orange with maximum shoot proliferation rate (99.75%), shoot number per explant (3.10), shoot length (10.70 cm), leaf number per explants (12.50) after three weeks of culture. In all experiments, no growth was observed for the basal MS medium. Phytohormones combinations of indole-3-acetic acid, IAA (1.2 mg/L), and kinetin (2.0 mg/L) were found to be the best for shoot proliferation. Among the cultivars, significantly, the highest rooting rate (81.25%), root number (4.95), and root length (2.95 cm) were recorded for Washington naval orange cultivar. The least rooting rate (48.45%), root number (3.55), and root length (2.26 cm) were observed from the Valencia cultivar. Significantly, the highest rooting rate (84.90%), root number per microshoot (5.20), and root length (3.05 cm) for MS medium supplemented with 1.5 mg/L 1-naphthalene acetic acid (NAA). The comparison of different concentrations of IAA and NAA on root induction of microshoots from nodal segments of sweet orange cultivars demonstrated NAA as the more effective hormone than IAA.

Screening of Cork Oak for Resistance to Phytophthora cinnamomi and Micropropagation of Tolerant Seedlings

Massive propagation of cork oak (Quercus suber) individuals tolerant to Phytophthora cinnamomi (Pc) is probably the most important challenge for cork production. Screening for resistance to Pc of ca. 200 seedlings obtained from a single cork oak tree that has survived the epidemic was performed by soil infestation. Twenty months after Pc inoculation, 33 seedlings survived from Pc infection and the four most vigorous seedlings were selected. The plants were forced to produce new shoots under controlled climatic conditions, and the new shoots were used to establish the plants in vitro by axillary budding. High axillary shoot proliferation rates were achieved by culturing the new shoots on Lloyd and McCown (WPM) medium, followed by subculturing for 2 weeks on 0.22 µM benzyladenine (BA) and for 2 weeks further on 0.04 µM BA. Addition of 20 µM silver thiosulphate (STS) increased the proliferation rates and improved the appearance and development of shoots. Rooting rates of 80–100% were obtained by culturing the shoots for 24 or 48 h on Gresshoff and Doy medium with ⅓ macronutrients plus 122.5 µM indole-3-butyric acid and subsequent transfer to root expression medium containing 20 µM STS. The results of this study optimize the micropropagation of a relevant and recalcitrant tree species in forestry.

In Vitro Propagation of Philodendron erubescens ‘Pink Princess’ and Ex Vitro Acclimatization of the Plantlets

This study describes the in vitro propagation and ex vitro acclimatization of Philodendron erubescens pink princess, one of the most popular ornamental variegated foliage plants. For shoot proliferation, the protocorm-like bodies of the Philodendron pink princess were cultured on solid Murashige and Skoog (MS) media supplemented with 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) at different concentrations. The results revealed that supplementation with BAP alone at a concentration of 1.0 mg/L yielded the maximum number of shoots and leaves. Furthermore, the application of BAP at 1.0 mg/L significantly enhanced the shoot proliferation of Philodendron pink princess when grown in liquid MS medium, yielding 11.2 shoots/explant and 4.7 leaves/explant. When the established microshoots were subjected to root induction using solid MS media supplemented with different kinds and concentrations of auxins, indole-3-butyric acid (IBA) at 3 mg/L resulted in the highest number of roots (3.2 roots/explant) and longest root length (1.9 cm). Three supporting materials, i.e., peat moss, vermiculite, and perlite, were used as planting media for the ex vitro acclimatization of the Philodendron pink princess plantlets. The results demonstrated that the in vitro plantlets acclimatized and exhibited a relatively high survival frequency in all planting media without morphological abnormalities. Peat moss outperformed all other types of planting media in terms of sustaining the vegetative growth of the plantlets. In the future, the approach established in this study could be employed for the extensive production of Philodendron pink princess.

In vitro direct organogenesis of the medicinal single-mountain local prioritized vulnerable Greek endemic Achillea occulta under different medium variants

Achillea species are of high medicinal value based on their use in traditional medicine and their content in secondary metabolites with potential application in dermatology and cosmetic industry. Achillea occulta is a single-mountain local prioritized vulnerable Greek endemic encountering propagation problems through seeds and root division. In vitro propagation can be used for the rapid and mass production of clonal plants complying with phytochemical and sanitary quality criteria. In this study, several culture medium variants were tested including the effect of different indole-3-butyric acid (IBA) concentrations with 6-benzyladenine (BA), different cytokinin types, agar concentrations, and zeatin (ZT) sterilization methods on in vitro shoot proliferation, as well as the effect of different auxin types and concentrations on in vitro rooting and ex vitro survival rate of shoot-tip explants. The results showed BA was the most preferable cytokinin type as evidenced by all proliferation attributes and autoclaved ZT provided longer shoots. Therefore, the two more promising and cost-effective treatments for proliferation were 2.2 μM BA + 0.25 μM IBA or 4.4 μM BA + 0.5 μM IBA after 21 days of culture on modified Murashige-Skoog (MS) medium (x2 Fe) enriched with 20 g L-1 sucrose and 6-7 g L-1 Agar (90-93.8% shoot formation, 2.7-2.9 shoots 0.4-0.6 cm long, 2.5-2.6 proliferation rates). Explants rooted better after 21 days of culture on plain MS medium with 2 μM IBA or 1 μM α-naphthalene acetic acid (NAA) (13.3-20% rooting, 1-1.5 roots 1.8-2 cm long). NAA gave more roots, IBA longer roots, while both auxins exhibited highest rooting (13.33-20%) and ex vitro survival rates (50-66.7%).

Micropropagation of strawberry (Fragaria ananassa “pajaro”)by meristem culture method

Experiments were carried out to examine the effects of different combinations of plant growth regulators in vitro micropropagation of strawberry plants. Specimens were sterilised by a different concentration of sodium hypochlorite - NaOCl (1, 2, and 3%) for 20 mins. A meristem was separated and cultured on Murashige and Skoog (MS) containing 0.3 mg/l BAP for 8 weeks. Initial shoots were transferred to MS with various concentrations of BAP (0, 0.1, 0.3, and 0.5 mg/l) and kinetin (0, 0.1, 0.2, and 0.3 mg/l) in either single or combination. After 6 weeks of culture, the regenerated shoots were transferred on 1/2 MS with different concentrations of NAA (0, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/l) and BAP (0, 0.1, 0.3, and 0.5 mg/l) in either single or combination. The results showed that the lowest percentage of contamination (10%) and the highest percentage of regeneration (80%) response were achieved on MS media by sterilisation with 3% NaOCl for 20 mins. The highest shoot proliferation per explant (16.2 shoots/explant) was reached on MS with 0.3 mg/l BAP plus 0.1 mg/l kinetin. 1/2 MS media with 0.1 mg/l NAA plus 0.1 mg/l BAP induced 14.4 roots per shoot and produced the longest roots (9.1 cm). However, the higher concentration of NAA (0.3-1.0 mg/l) or in combination with BAP resulted in callus formation. The plantlets, thus developed, were hardened and successfully established in soil containing organic fertiliser: Trichoderma: coconut peat (1:1:1 v/v/v).

Morpho-Physiological Evaluation of Solanum betaceum Cav. In Vitro Cloned Plants: A Comparison of Different Micropropagation Methods.

Tamarillo (Solanum betaceum Cav.) is a subtropical solanaceous tree with increasing agronomic interest due to its nutritious edible fruits. Growing demand for tamarillo plants and fruits requires optimization of existing propagation methods and scaled-up systems for large-scale cloning of selected germplasm. Three in vitro protocols have been used to micropropagate tamarillo: (1) axillary shoot proliferation in a semisolid medium, (2) organogenesis, and (3) somatic embryogenesis procedures. Variables such as the age of the established shoot cultures and rooting treatments were also analyzed. The morphological and physiological quality of acclimatized plants derived from all the methodologies were compared, with seed-derived plants used as a control group. Overall, the results show that in vitro-derived plants have a similar development to seed-derived plants. Micropropagation by axillary shoot proliferation was highly efficient, with rooting rates above 80% in most treatments. Organogenesis induction was more effective from lamina explants using MS media with 2.0 mg·L-1 6-benzylaminopurine. Both organogenesis and somatic embryogenesis-derived plants were also morphologically and physiologically equivalent to seed and axillary shoot-derived plants. The specificities of each micropropagation method are discussed.