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Low temperature studies with winter-dormant buds are severely limited by the lack of a rapid,non-destructive assay for their viability. Investigations involving the winter harvest of ecodormant buds of woody subjects, including cryopreservation, are restricted if viability cannot be assessed until dormancy is broken. If post-treatment grafting indicates low survival of the harvested population then further collection and study has to be delayed until the next winter season. This study trials the use of a portable gas exchange system able to discriminate between live and dead buds rapidly, with the assay confirmed as non-destructive by subsequent micropropagation. Active respiration was recorded for 85% of a winter-dormant Malus domestica buds population that showed 91% viability when grafted (n = 45). Lethally stressed material gave no false positive results. When micropropagated after respiratory measurement, a population viability of 76% was recorded. There was a significant, positive correlation between respiration and fresh weight for buds of mass >10 mg, from a population with a mean fresh weight of 17 mg.

As in vitro culture environments can be mutagenic, it's challenging to produce true-to-true type plants from meristems. Therefore, the present study aimed to devlop an efficient protocol for micropropagation from shoot tip explants of medicinally important Aloe vera using different concentrations of A. vera leaf gel (AvG) as organic supplement to the culture media. Assessment of the genetic stability of A. vera in vitro multiplied plants compared to the donor plant is one of the most important pre-requisites especially before its use for commercial and medicinal purposes. For shoot multiplication, the highest number of axillary shoots recorded was 13.27 ±5.11 with an average length of 2.21 ±0.84 cm on M3 medium (MS + 0.2 mg L−1 of β-indole butyric acid (IBA) + 3 mg L−1 benzylamino-purine (BA) + 5% AvG). All three strength of MS medium (1, 1/2, 1/4) with different concentrations of AvG resulted in 100% root induction after 4 weeks of culture. All the rooted microshoots (100%) were successfully acclimatized. To provide a reliable conclusion on its nutritive supply to MS medium, the total phenolic (TPC), condensed tannins (TTC), flavonoid (TFC), and flavonol (TFlC) content as well as the antioxydant activity of AvG were investigated. TPC and TTC values were found to be 31.02 ± 0.46 μg gallic acid equivalent mg−1 of extract and 4.00 ± 0.96 μg catechin equivalent mg−1 of extract, respectively while TFC and TFlC were 13.58 ± 0.84 and 9.75 ± 0.58 μg rutin equivalent mg−1 of extract, respectively. The results of antioxydant activity of AvG following DPPH, FRAP and ABTS assays were equal to 12.50 ± 0.89; 49.59 ± 2.07 and 64.93 ± 3.23 µmol Trolox equivalent mg−1 of extract, respectively which may explain the positive role of AvG as supplement. The genetic stability of in vitro propagated A. vera plants was evaluated using 10 inter simple sequence repeat (ISSR), 10 start codon targeted (SCoT) and 10 simple sequence repeat (SSR) markers. Out of these, 5 ISSR, 3 SCoT and 6 SSR primers produced resolvable, reproducible and scorable bands. ISSR primers generated 32 amplification products ranging from 200 to 1040 bp in size with a mean number of 6.4 bands while SCoT primers generated 33 amplicons with an average of 11 bands in the range of 160–2410 bp. In the SSR products, 6 bands were observed, each primer produced one band ranging from 410 to 720 bp. All banding profiles were monomorphic proving that the stability of the micropropagated A. vera plants was maintained. This study proved the suitability of supplementing in vitro culture media with AvG to enhance direct regeneration through shoot tip explants of A. vera for large scale micropropagation applications.

This study represents an efficient preliminary protocol for in vitro mass production of two Paulownia species (Paulownia hybrid and Paulownia tomentosa) seedlings by using seed explant. Different concentrations of benzyladenine (BA) or Kinetin (Kin) (0.0, 2.0, 4.0, 6.0, 8.0 and 10.0 mg/L) were tested during multiplication stage. The number of shoots/explants was significantly increased with increasing either BA or Kin concentration; however, the shoot length significantly decreased. Data show that media fortified by BA (10 mg/L) combined with indole butyric acid (IBA) at 1.0 or 1.5 mg/L recorded the highest number of shoots/explant (9.13 and 9.25, respectively). After six weeks during the multiplication stage, data cleared that media fortified by benzyladenine (10 mg/L) combined with IBA at 0.5 mg/L recorded the highest shoot length (3.23 cm). The inclusion of indole butyric acid (IBA) or naphthalene acetic acid (NAA) at 1.0–1.5 mg/L to the medium significantly increased the number of roots/plantlets and the highest root length. The results indicated that IBA supplementation was more effective than NAA for in vitro rooting of both Paulownia species. The best treatment for multiplication was 10 mg/L and 8.0–10 mg/L BA for P. hybrid and P. tomentosa, respectively. Peat moss and sand (1:1, v/v) or peat moss and sand (1:2, v/v) were investigated as soil mixture during the adaptation stage. The results referred that Paulownia species plantlets were successfully survived (100 %) in soil mixture contained peat moss: sand (1:2, v/v). This mixture recorded the highest values of plantlet height and number of leaves/plantlets.

Three species of Begonias endemic to Java and Sumatra, namely Begonia leuserensis, Begonia atricha and Begonia scottii, were conserved in Cibodas Botanic Gardens as sources of germplasm for ornamental plant and/or medicines. However, the information on efficient hormones concentration and their culture media application through an in vitro propagation effort is still limited. Therefore, this study aimed to explain the growth response of three species of Begonias using various hormones concentrations and culture media through in vitro propagation. The culture media using Murashige & Skoog (MS) media that combinedwith 6-Benzyladenine (BA) dan Thidiazuron (TDZ) hormones in different concentrations i.e. 0.5 mg/L, 1 mg/L, 2 mg/L, and 3 mg/L. Observation parameter included shoot number, plantlets height, and leaves number. The data were analyzed using analysis of variance (ANOVA) with the F test at a 5% significance level. The results showed that three species of Begonias were observed to have different growth responses in the combination of MS+BA and MS+TDZ media. The combination of MS+TDZ media produces more shoots number, while the combination of MS+BA media influenced higher in leaves number. A concentration of 0.5 mg/L of hormone showed a good regeneration, therefore were recommended for in vitro propagation of Begonia species.

Dalbergia latifolia Roxb. (Fabaceae), famed for its wood, is a vulnerable plant species according to the International Union for Conservation of Nature and Natural Resources (IUCN) Red List as a result of its over-utilisation. This mini-review, which provides a synthesis of the micropropagation of this woody plant species, can be useful for the establishment of a more focused and comprehensive micropropagation protocol. An in-depth analysis of previously published reports indicates that the most commonly employed explant in D. latifolia in vitro regeneration protocols are seedling-derived explants such as cotyledonary nodes since the use of ex vitro material leads to considerable contamination and phenolic oxidation. The media composition for culture initiation, multiplication, maintenance and rooting has been thoroughly studied but in most studies, pertinent information about acclimatization and plant survival in the field was not provided, reducing the reproducibility of protocols. This mini-review provides possible solutions to overcome explant contamination and browning during D. latifolia in vitro culture establishment.

Nepenthes khasiana Hook. f. is a critically endangered, carnivorous, endemic species of the Assam and Meghalaya and belongs to the monotypic family Nepenthaceae. An efficient micropropagation protocol was optimized for the propagation and ex-situ conservation of elite germplasm of the species from axenic shoot tip culture. The axenic cultures were established through in vitro seed germination, which envisage the prolonged seed germination time (180 Days) and poor seed germination (25%) in the species. Application of GA3 (500 ppm) enhanced the seed germination upto 89%. Half-strength Murashige and Skoog (MS) medium fortified with 4.4 µM BAP was reported to be the ideal medium and 96.81% shoot formation was observed in N. khasiana shoot tip explants. The highest shoot proliferation of 21.22 ± 0.78 shoots per explants with 10.5 ± 0.5 cm mean shoot length was recorded. The in vitro raised shoots rooted in half salt strength MS medium fortified with IBA (4.9 μM) and 92.11% rooting was recorded with average 17.58 ± 0.87 roots per shoot and 5.5 ± 0.29 cm root length. In-vitro raised plantlets were shifted to the glass house with 65% success rate. Thus, the protocol developed might be useful for the ex-situ conservation of germplasm and commercialization of species to cope up with the ever increasing medicinal and ornamental demands of the species.

Background: Plant tissue culture involves the use of explants obtained from plants to induce organogenesis with the help of plant growth regulators (PGRs). Micropropagation techniques provide a faster and economical solution to the limitations associated with traditional methods of plant cultivation. The present study focuses on the multiple shoot induction and proliferation of Ficus carica var. Black Jack. Factors that influence the growth of in vitro multiple shoots on the apical buds, which include growth media and PGRs, were investigated in this study. Different concentrations of cytokinins like 6-benzylaminopurine (BAP), Thidiazuron (TDZ), and Kinetin (Kin) were used on woody plant medium (WPM) for the optimization of media for multiple shoot induction and proliferation. Results: Apical buds of Ficus carica var. Black Jack growing in WPM supplemented with BAP produced the healthiest plantlets, with the highest number of multiple shoots. The most efficient medium composition which produced the highest number of multiple shoots (37.8) per growing explant was WPM supplemented with 20 µM BAP. Proliferated multiple shoots were efficiently rooted using WPM + 20 µM BAP + 8 µM indole-3-acetic acid (IAA). This optimized medium composition significantly enhanced the production of multiple, disease-free plantlets using single apical bud explants of Ficus carica var. Black Jack. Conclusions: In the present study the observations indicate that WPM supplemented with 20 µM BAP is the best-suited medium for organogenesis and multiple shoot culture of Ficus carica var. Black Jack, and this technique can be potentially applied for commercialization of the plant. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

Plantlets of Hemidesmus indicus (L.) R. Br. (Indian sarasaparilla) were developed in vitro in two different types of culture vessels and closure types—(1) culture bottles with polypropylene screw-caps and (2) conical flasks with cotton plugs both containing Murashige and Skoog medium with 1.0 mg L−1 6- benzylaminopurine, 0.5 mg L−1 kinetin, and 0.1 mg L−1 indole-3-acetic acid. Shoots proliferated in the bottles exhibited production of (i) a few leaves with reduced leaf area, (ii) wiry and brittle stem, and (iii) less number of shoots, whereas, the shoots developed in culture flasks were of superior characteristics with healthy leaves. The leaves developed in bottles revealed the presence of under-developed cuticle, lower palisade/spongy ratios, undifferentiated epidermal layers, abnormal and non-functional stomata, absence of periderm, and unorganized ground and vascular tissue systems preceded the development of hyperhydrated shoots. The use of a conical flasks culture system promoted the development of natural anatomical features in plantlets. Overall, the type of culture vessel remarkably influenced the growth and development of in vitro-derived plantlets of H. indicus which could be explored for commercial propagation of this multipotent medicinal plant. Compared with glass bottles, cotton plugged flasks improved shoots formation, morpho anatomical parameters, rooting, and survival of in vitro plantlets of Hemidesmus indicus.

Rheum emodi Wall. ex Meisn is an important medicinal herb used in treating type II diabetes mellitus, arthritis, inflammation, and fever. Roots and rhizomes of this plant possess anthraquinones showing anticancer, antiviral, anti-inflammatory, anti-ulcerogenic, immunosuppressive, and pro-apoptotic activities. In the present study, in vitro propagation and adventitious root establishment were achieved for the detection and quantification of important secondary metabolites. In micropropagation experiments, high rate of callus induction was obtained from mid rib (100%) as well as leaf explants (97.67 ± 1.20%). The highest percent shoot induction was observed from leaf explants (76.25 ± 2.39%) as compared to callus (66.67 ± 2.20%). Regenerated shoots showed 70% rooting response with healthy long roots (5.73 ± 0.15 cm). Adventitious root culture response from nodal and leaf explants was better in liquid medium (93.33 ± 1.67% and 73.13 ± 2.87%) compared to semi-solid medium (85.0 ± 2.89% and 76.67 ± 3.33%). The highest DPPH (2–2 diphenyl, 1-picrylhydrazyl) free radical scavenging activity (92.73 ± 0.07%) and total antioxidant activity (6.24 ± 0.07 µg ascorbic acid equivalent (AAE) mg−1) was recorded in in vitro roots. Maximum phenol content (14.70 ± 0.29 µg gallic acid equivalent (GAE) mg−1) was present in adventitious root culture whereas flavonoid content (82.37 ± 0.12 µg quercetin equivalent (QE) mg−1) in in vitro shoots. The identification and quantification of secondary metabolites in in vitro shoots, in vitro roots, and adventitious root cultures were evaluated via high-performance liquid chromatography-mass spectroscopy (HPLC–MS) analysis which revealed the presence of three anthraquinones, emodin, physcion, and chrysophanol, in adventitious root cultures. HPLC–UV detector analysis showed the highest emodin concentration (2.24%) in in vitro shoots. The present study gives a highly reproducible micropropagation protocol for R. emodi as well as provides an alternative source of anticancer compound, emodin, from in vitro shoots and root cultures.

Turbinicarpus mombergeri is a cacti species formed by a hybridization process between Turbinicarpus laui and Turbinicarpus pseudopectinatus. Under natural conditions, it is very difficult for two species be genetically compatible for hybridization, and to produce flowers at the same time. Thus, T. mombergeri is a very interesting and a rare species. Unfortunately, the current populations are decreasing and now it is considered critically endangered. The aim of this research was to develop a successful protocol for propagating T. mombergeri using the in vitro culture techniques. Seed disinfection was performed with Plant Preservative Mixture, and 80% of germination occurred at day 45 in Murashige-Skoog medium. The shoots were cut longitudinally, and the segments were transferred to media containing 2.22 or 4.44 µM benzyladenine to induce shooting. The generated shoots were highly hydrated, and presented abundant callus. The hyperhydricity was controlled by reducing salt medium concentration, by increasing calcium levels and by using polyethylenglycol. The reduction of callus was attained by adding tri-iodo benzoic acid. Vigorous and thick shoots were generated in medium containing urea, and rooting improved in the presence of 0.5 µM indoleacetic acid. Plantlets with normal morphology were obtained, and the survival rate of the plants in soil was 80%. The methodology developed represents an alternative for propagation of T. mombergeri under controlled conditions for commercial or conservation purposes. The paper describes several approaches to avoid the hyperhydricity and callus formation, to improve quality of shoots and to increase the development of roots during the micropropagation process of the endangered cacti T. mombergeri.

Micropropagation through nodal explants was attempted for a rare and endemic taxon Corynandra chelidonii var. pallae (Cleomaceae) in view of its medicinal uses, bioactive compounds, declining natural populations due to intermittent sterility, low seed set and erratic seed germination. Multiple shoots were regenerated directly from the nodal explants on Murashige and Skoog’s medium containing different concentrations and combinations of cytokinins and auxins. High-frequency multiple shoots of 8.15 ± 0.249 with 1.63 ± 0.031 shoot length (cm) were obtained on MS medium augmented with the combination of 0.5 mg/L of thidiazuron and 1.0 mg/L of indole-3-acetic acid. Directly regenerated shoots were rooted with 95% root induction on a half-strength MS medium supplemented with 1.0 mg/L of indole-3 butyric acid. The in vitro raised plantlets were hardened in plastic pots and acclimatized initially in the greenhouse conditions and later transferred to the field conditions with a 78% of survival rate. The genetic fidelity of in vitro generated and field transferred plantlets with that of the mother plant was checked by carrying ISSR marker-based polymerase chain reaction method. This protocol on direct organogenesis and genetic fidelity analysis in Corynadra chellidonii var. pallae can be successfully employed for large-scale multiplication and conservation.

In this study, a new method for shoot regeneration via protocorm-like bodies (PLBs) is described. Shoot proliferation using stem elongation under light-emitting diodes (LEDs), and enhanced growth of Anthurium andraeanum ‘Tropical’ plantlets on medium containing silver nanoparticles (AgNPs) was investigated. For shoot regeneration, the micropropagation efficiency via PLB formation was fourfold more efficient than directly from callus after 12 wk of culture. This was possible because each initial callus cluster could be cut into 20 explant slices (1 × 5 × 5 mm in size), each with an average of 42.67 PLBs which are capable of growing into plants. For shoot proliferation, single shoots with 2 cm in size derived from PLB culture were placed under different blue to red LED ratios. This study showed that higher shoot height (4.77 cm), fresh weight (327.33 mg), number of leaves per shoot (6.33), and the number of stem nodes per shoot (5.67 stem nodes) were obtained under 70R:30B as compared to those under other lighting conditions and fluorescent lamp after 8 wk of culture. Especially, the total shoot proliferation (10 shoots) via stem node culture was higher than those in the control. For enhanced plantlet growth, single shoot with a pair of leaves derived from stem elongation under 70R:30B cultured on 1/4 MS medium supplemented with 0.5 mg L−1 NAA and 1 g L−1 activated charcoal, 7 mg L−1 AgNPs, 30 g L−1 sucrose, and 8 g L−1 agar gave the highest growth than other treatments. Plantlets derived from 7 mg L−1 AgNP treatment transferred into greenhouse showed better growth as well as 4-wk earlier flowering than the control treatment.

The objective of this work was the vegetative rescue of Cedrela odorata L. adult trees from forest areas by micropropagation using shoot regeneration from axillary buds in vitro. Nodal segments (0.5 and 1.0 cm) of shoots formed from cuttings taken from canopy sprouts were used. The explants were successfully established in vitro on MS medium supplemented with 2.22 ?M BAP, 0.49 ?M AIB, 0.28 ?M GA3 and PPM (0 and 8.6 ?M). Shoot multiplication on MS with combinations of GA3 (0.28 and 1.12 ?M) and BAP (2.2, 3.3, 4.4, 6.6, and 8.8 ?M) and shoot rooting on medium with 4.9 ?M AIB, 5.7 ?M AIA or 5.37 ?M ANA were evaluated. The MS medium supplemented with 0.28 ?M GA3 and 8.8 ?M BAP generated 4.16 shoots per explant. The best rooting induction was observed on medium containing 4.9 ?M IBA, resulting in 60% of shoot rooting. The plantlets rooted were acclimatized and showed normal development with 97% survival rate. The use of canopy sprouts as explants is feasible in the rescue of C. odorata and the combination of BAP and GA3 favors in vitro multiplication.

Jatropha is a non-edible, important bioenergy plant, which can grow in marginalized land. The seeds possess about 36% oil and this would be converted into biodiesel or biojet-fuel. Jatropha provides an option for sustainable feed and fuel production due to its inherent qualities including hardy nature, drought tolerance and surviving with limited amount of water, tolerance to unfavorable conditions and excessive moisture. However, heterozygosity, low productivity and poor understanding of its genome are the major impediments to elite line development. Further, classical breeding and advanced technological investments remain limited owing to log juvenile phase and breeding cycles. Scientific technologies that lead to identification of elite genotypes and development of high yielding elite Jatropha lines and effective methods of detoxification of seeds needs immediate priority. Efficient tissue culture system, doubled haploids (DH) and genomic tools are increasingly made available to improve the seed yield and its oil quantity through the development of geminivirus disease resistant lines. The application of advanced, sequencing technologies has presented a repertoire of genomic information for this important yet orphan crop. In the present investigation, we highlight the achievements made in Jatropha towards development of high yielding, virus resistant elite lines and hybrids with yield potential ranging from 3 to 5 tons per hectare in a year, which is a first ever report in the world. We also developed potential biotechnological tools such as genetic transformation, genome editing and next generation genomics tools including linkage maps and QTLs for accelerating breeding efforts through marker assisted selection. Because of our concerted and continuous efforts during the past 15 years, we have overcome all the obstacles and developed high yielding, disease resistant hybrids/lines, advanced cultivation technologies with thorough knowledge on Agronomy of the Jatropha crop. The Jatropha cultivation technology developed for the first time in the world, could open up new avenues for higher yield productivity of commercial viability.

Micropropagation protocol of Oriental Hybrid Lilium cv. Ravenna was developed using bulb scale segments (Basal and Tip) as explants. Surface sterilization of healthy bulb scales with carbendazim 200 ppm for 30 min, then 0.1 percent mercuric chloride for 10 min, then 70% ethyl alcohol for 30 s was superior to all other treatments in recording highest culture asepsis (77.08%) and higher explant survival (86.12%). Explant survival was higher in basal segments (88.54%) compared to tip segments (85.52%). Highest culture establishment was recorded in basal scale segments (68.26%) followed by tip scale segments (55.21%). MS medium augmented with 0.50 mgl−1 Naphthalene acetic acid and 2.0 mgl−1. 6-Benzylamino Purine recorded maximum culture establishment (76.17%), highest bulblet number/explant (5.52) with maximum length of shoots (2.20 cm) and number of leaves (3.39). This treatment combination of growth regulators resulted in highest shoot proliferation (83.33%) along with maximum shoot number (2.41explant−1), shoot length (2.35 cm) and leaf number (5.44) of micro shoots during proliferation stage. Rooting of explants was superior with Indole-3-butyric acid compared to Naphthalene acetic acid. Highest rooting of 92.71% along with maximum number of primary roots shoot−1 (12.06), maximum primary root length (3.17 cm) was documented in Murashige and Skoog medium added with Indole-3-butyric acid 1.50 mgl−1 with best ex vitro survival rate (98.96%) of rooted plantlets during primary hardening in perlite + vermiculite (1:1) mixture.

Jatropha is a drought-tolerant plant producing seed oil for the biodiesel. Limitation to the development of jatropha is unavailability of high-yielding varieties and efficient in vitro regeneration system which is required for micropropagation. In this study, in vitro regeneration system from jatropha juvenile cotyledon was established. Firstly, concentrations of hormones in the MS medium were optimized and it was found that 1.5 mgL-1 benzyl adenine (BA) + 0.05 mgL-1 indole-3-butyric acid (IBA) + 0.5 mgL-1 thidiazuron (TDZ) turned out to be the best for shoot induction (3.82 ± 0.18 shoots/explant). Secondly, shoot induction medium was fortified with different concentrations of glutamine and adenine sulfate. It was found that 25 mgL-1 each of glutamine and adenine sulfate was the most effective, resulting to 9.09 ± 0.37 shoots/explant and 93.0% regeneration frequency. Regenerated shoots were cultured on medium containing 0.5 mgL-1 BA and different concentrations of gibberellic acid (GA3), 0.5 mgL-1 GA3 with 0.5 mgL-1 BA were found to be the best for shoot elongation (2.13 ± 0.18 cm). The highest frequency of root (40%) was observed on the medium with 0.5 mgL-1 IBA. The established procedures will be useful for the mass propagation and genetic transformation of elite jatropha genotypes. Key words: Adenine sulfate (Ads), benzyl adenine (BA), indole-3-butyric acid (IBA), in vitro regeneration, glutamine (Gln).

Hoya imperialis (H. imperialis) and H. coronaria (Apocynaceae) are known to have ornamental value due to their beautiful flowers; however, the feasibility of propagating these plants have not been reported despite the wild populations in Brunei Darussalam being highly threatened due to habitat loss and overcollection. Thus, the present study aimed to conduct a preliminary study of the feasibility of two alternative propagation methods, stem cutting and micropropagation, as a potential approach for their ex situ conservation. Hoya stem cuttings were treated with either indole-3-butyric acid (IBA) or 1-naphthaleneacetic acid (NAA) (0–2000 mg/L), and then propagated onto a mixture of peat moss and perlite. For micropropagation, Hoya leaf explants were cultured onto Murashige and Skoog (MS) agar media that were supplemented with IBA and/or kinetin (KN) (0–10.0 mg/L). This present study shows that both Hoya species were successfully propagated by stem cutting even without hormone treatment. However, interestingly, in H. imperialis, when compared with control, the mean number of new leaves (6.3 ± 1.0) and the mean relative growth rate (RGR) based on stem diameter (0.004 ± 0.0007 cm cm−1 day−1) significantly increased when treated with 500 mg/L NAA and 2000 mg/L IBA, respectively. Meanwhile, in H. coronaria, significantly higher mean number of roots was achieved by treating with 1000 mg/L NAA (16.6 ± 1.4) or 2000 mg/L IBA (17.5 ± 2.7) compared with control. For micropropagation, callus induction was not promising and could only be observed at specific concentrations of both IBA and KN, with H. imperialis appearing to be more responsive towards these hormones in comparison to H. coronaria. The present study showed that stem cutting appeared more feasible in propagating both Hoya species.

In this study, an efficient method for in vitro regeneration of Plumbago europaea was developed using direct and indirect organogenesis. Accordingly, micropropagation and regeneration were obtained on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators. The effects of explant type and plant growth regulators on shoot organogenesis of P. europaea were evaluated. For the nodal explants, MS medium containing 0.5 mg/l TDZ (11.62 shoots per node) was the best medium for high frequency of micropropagation. In comparison, the highest percentage of direct organogenesis (70%) and number of shoots per explants (14.6) were acquired for the internode explants using 0.5 mg/l TDZ and 0.1 mg/l IAA. The obtained data revealed that TDZ is the most effective cytokinin for the direct shoot organogenesis. The highest indirect organogenesis rate was observed using 2 mg/l BA and 0.1 mg/l NAA for the internode explant. The maximum number of roots was distinguished on ½ MS medium containing 0.5 mg/l IBA (6.42). The rooted plantlets were gradually hardened and acclimatized under ex vitro conditions. As an important outcome, the active compound plumbagin was found mainly in the root tissues of the micro-propagated and regenerated plantlets. Taken all together, this study achieved a successful protocol for in vitro regeneration of P. europrea and could be considered for large-scale multiplication of this important medicinal plant.

Plant micropropagation comprises biotechnological tools used for the conservation and mass propagation of orchid species. However, there are few reports of its use for orchids of the subtribe Pleurothallidinae. The present study evaluated the effects of 6-benzylaminopurine (BAP) on shoots formation and development of Dryadella zebrina (Porsch) Luer. For this, seeds were in vitro germinated, and the plantlets were submitted to different concentrations of BAP (0, 3, 6, 9, 12, and 15 µM). The plantlets derived from the treatments 0, 6, and 15 µM of BAP were collected after 60 days in culture and subjected to light microscopy analysis. Our results indicated that BAP increased the formation of the new shoots of D. zebrina, especially in treatment with 6 µM, and its use is indicated for the in vitro multiplication phase. The anatomical analyses of the roots showed a deleterious effect of 15 µM BAP on the meristematic region, with the presence of more vacuolated cells in this zone. Our results represent the first successful report of in vitro propagation for the genus Dryadella and may serve as a basis for further studies of in vitro propagation of phylogenetically related species.

Alangium salviifolium (L.f.) Wangerin, sage-leaved alangium, is a medicinally important tree species of the Alangiaceae. It is a source of numerous bioactive therapeutic compounds, but its conventional propagation through seeds is beset with poor germination and seedling survival. Here, we report an efficient and reproducible protocol for its micropropagation using nodal segments and shoot apices collected from two-month-old plants. Murashige and Skoog (MS) medium and Woody Plant Medium (WPM) supplemented with various concentrations of plant growth regulators, either alone or in combination were evaluated. Bud break (≈ 71%) occurred from nodal explants after 3 weeks on WPM containing 1.11 µM N6 -benzyladenine (BA) plus 2.64 µM α-Napthoxy acetic acid (NAA), producing a single shoot of about 1.82 cm long by 4–6 weeks. WPM containing 1.42 µM IAA and 1.11 µM BA was optimum for shoot proliferation from nodal explants and shoot apices, yielding an average of 2.80 shoots per nodal explant after three successive subcultures. Elongated shoots were separated from the clumps and multiplied. Once they were 2.5–3.5 cm tall they were transferred to WPM medium with 2.46 µM indole-3-butyric acid (IBA), where 53–60% of shoots developed 1–3 cm long roots within 10–15 days. A reverse photoperiod (16 h dark/8 h light) was crucial for root induction. Well rooted shoots were transferred to pots, acclimatized in greenhouse and 60% of plants were transferred to soil after hardening. This is the first report of micropropagation in A. salviifolium and this protocol could serve for large scale propagation of this medicinal tree species. The effects of auxins and cytokinins on shoot multiplication and rooting of the medicinal tree Alangium salviifolium (L.f.) Wangerin were studied. A reverse photoperiod enhanced rooting. Plants survived field transfer.

Ammonium nitrate (AN) is one of the major nitrogen sources of Murashige and Skoog (MS) medium. It is prohibited in various countries, including India because it is used in explosive manufacturing. Since MS is the most successful medium used for in vitro culture of many plant species, an attempt was made to achieve the composition of MS medium using ammonium hydroxide and nitric acid as an alternative to AN. This acid–base neutralization product AN, was further characterized by ATR-FTIR spectroscopy. Micropropagation of Musa acuminata cv ‘Grand Naine’ was tested using the alternate MS medium (AMS) and good mean shoot number was achieved. Shoot proliferation of M. acuminata cv ‘Grand Naine’ on AMS was significantly better than on normal MS medium. A 1-year production cycle of M. acuminata cv ‘Grand Naine’ was successfully accomplished with seven successive subcultures and rooting on AMS medium followed by satisfactory acclimation. To check broad cross-species utility of AMS for shoot proliferation, a range of species including Chlorophytum borivilianum, Dalbergia sissoo, Dregea volubilis and Plumbago zeylanica were tested. The in vitro shoot multiplication rate of these species on AMS was statistically not different from MS medium. These results indicate that AN can be replaced with ammonia hydroxide and nitric acid in preparing MS-based medium, without negatively affecting shoot proliferation/ rooting and it would be cost-effective too for micropropagation operations in comparison to commercially available MS medium. Commercial NH4NO3 could be replaced with HNO3 and NH4OH in tissue culture medium to follow the explosive regulations and was efficient for in vitro culture of various plant species.

Hibiscus moscheutos L., also known as hardy hibiscus, is valued for its medicinal and ornamental attributes. It is usually propagated via seeds or cuttings. The purpose of this investigation was to develop a dependable micropropagation for H. moscheutos ‘Luna White’. To that end, the effect of four explant types (leaf, root, node, shoot tip) and two growth regulators 6-benzylaminopurine (BA) and meta-Topolin (mT) (6-(3-hydroxybenzylamino) purine) on in vitro growth of H. moscheutos was investigated. Genetic stability of the in vitro grown plants was assessed using flow cytometry, and chromosome count was investigated. No shoots were obtained from leaf or root explants. An efficient protocol for micropropagation of H. moscheutos using two explant types, 2-node and shoot tip explants, and two cytokinins (BA and mT) capable of producing true-to-type regenerants was established. Both BA and mT can be used at 2 μM or 4 μM using either 2-node or shoot tip explants. No significant difference was found between the nuclear DNA contents of seed-derived and in vitro grown plants (P < 0.05). The mean 2C DNA and monoploid 1Cx-values of seed-derived plants were 3.25 ± 0.08 pg and 1.62 ± 0.04 pg, respectively, compared with 3.26 ± 0.06 pg and 1.63 ± 0.02 pg, respectively, for in vitro grown plants. The chromosome number of both seed-derived plants and regenerants was determined to be 2n = 2x = 38. The mature regenerants obtained were fertile and phenotypically similar to seed-derived plants.

Jasminanthes tuyetanhiae is one of the precious herbs and good for human health, discovered in the Da-Bia Mountain (Phu Yen province, Vietnam). The identification of the CTW species (species believed to be J. tuyetanhiae collected in 2019) by molecular indicators showed that this species belongs to the Apocynaceae family. High similarity (98.2%) in the ITS1-5.8SrRNA-ITS2 gene sequence of CTW when compared on Genbank with J. tuyetanhiae and genetic distance difference (1.3%) - in the limit of difference of the same taxon considered on matK sequence shows that CTW is likely to belong to this species. Surface sterilization of ex vitro explants with HgCl2 (0.1%) for 3 min gave the highest sterilization efficiency compared to the remaining treatments with the induction explant rate of 93.33%. BA (0.5 mg l−1) is the most optimal for effective in vitro shoot multiplication in this species. Adding IBA to culture media at the appropriate concentration (0.5 mg l−1) increased rooting ability, shortened rooting time (13 days) and increased root length (6.53 mm). The ventilation culture system improves rooting efficiency, stimulates growth and induces secondary root formation—which is a suitable system for in vitro propagation of the Jasminanthes tuyetanhiae in this study. Jasminanthes tuyetanhiae is one of the precious herbs and good for human health, discovered in the Da Bia mountain of Phu Yen province. High similarity (98.2%) in the ITS1-5.8SrRNA-ITS2 gene sequence of CTW when compared on Genbank with Jaminanthes tuyetanhiae. In vitro propagation is an effective method for the conservation and development of the Jaminanthes tuyetanhiae plantlets on a commercial scale.

In addition to various medicinal properties, Acorus calamus (sweet flag) is used in health, food, and perfume industries. Since this species is a rare plant in Iran, its propagation and cultivation are of the great importance. The aim of this study was to investigate the effects of different plant growth regulators on micropropaga-tion of this plant and to select the appropriate population. The root, the rhizome and the leaf explants of three populations (Arzefon, Pelesk, and Alandan) were cultured on MS medium supplemented with different concentrations of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP) for callus induction and plant regeneration. The results showed that only rhizome explant resulted in direct plant regeneration. Among different treatments, the 1 mg/l treatment of BAP and NAA - with the highest mean number of regenerated plants (3.75 ± 0.85), the highest percentage of grown explants (91.6%) and maximum average length of regenerated plants (12.06 ± 0.32 cm) - was the best treatment for regeneration of sweet flag. The highest mean number of root (6.6 ± 0.1) was observed in Alandan population in 1 mg/l treatment of indole-3-butyric acid (IBA). According to the present study, Alandan population is suitable for cultivation purposes in Iran.

The study reports a low-cost micropropagation protocol for Nardostachys jatamansi, a high value, critically endangered Himalayan herb. Shoot-bud bearing rhizome segments of the plant were made to sprout and elongate on 0.8% agar-gelled Murashige and Skoog's medium containing 3% sucrose. The sprouted shoots were then multiplied on agar-gelled as well as liquid MS medium containing various concentrations and combinations of kinetin (Kn) and 1-phenyl-3- (1,2,3-thiadiazol-5-yl) urea (thidiazuron or TDZ). Maximum number of shoots developed per explant after 30 days on 100 ml agar gelled (6.7 shoots) and 30 ml liquid (6 shoots) MS medium containing 0.5 mg l−1 Kn and 0.75 mg l−1 TDZ. Although rooting of in vitro shoots was attempted on full as well as half-strength MS medium containing different auxins, rooting was achieved in 100% shoots (3.07 roots/ shoot) after 30 days on half-strength MS medium supplemented with 1.0 mg l−1 indole-3- acetic acid (IAA). Different potting mixtures were used to acclimatize the micropropagated plantlets under polyhouse condition, but a maximum of 83% survival was recorded in sand and sand plus soil. The genetic fidelity of the in vitro plantlets was also assessed using Start Codon Targeted Polymorphism (SCoT) and Directed Amplification of Minisatellite DNA (DAMD) markers, and a total of 8.03% polymorphism was recorded. Eventually, the cost of micropropagation was reduced by 6.4 times during shoot multiplication and further by half during rooting. The present micropropagation method developed for N. jatamansi can have potential applications in medicinal plant-based industry.

An efficient micropropagation protocol was developed for C. asiatica, a commercially important herb having high medicinal and cosmetic properties. Shoot induction was observed on Murashige and Skoog (MS) medium fortified with various combinations of benzyladenine (BA) (2.22 - 22.2 µM) and naphthalene acetic acid (NAA) (1.34 - 8.06 µM). The maximum number of shoots (11.30 ± 0.68) per explant was initiated on MS media containing BA (6.66 µM) and NAA (2.69 µM). Maximum shoot proliferation was achieved on MS medium supplemented with BA (6.66 µM) and indole-3-acetic acid (IAA) (2.84 µM). The best rooting (24.69 ± 0.08) per explant was achieved on half-strength MS medium supplemented with indole-3-butyric acid (IBA) (4.92 µM). The in vitro regenerated plantlets were acclimatized and shifted to the field. Genetic stability of the micropropagated regenerants was evaluated using inter-simple sequence repeat (ISSR) markers and flow cytometry. Out of 28 primers tested, 9 primers resulted in 561 distinct and reproducible bands ranging from 200 to 1400 bp. Monomorphic banding profile was observed between the mother plant and tissue culture regenerants. The mean 2C DNA content of mother and micropropagated plants evaluated by flow cytometry were estimated to be 0.14 and 0.12 pg, thereby indicating no significant change in the 2C DNA content. Additionally, HPLC (High performance liquid chromatography) analysis did not show any significant differences in the content of triterpenes and phenolic compounds in both mother and micropropagated plants. Thus, this protocol could be implemented for large scale cultivation of elite germplasm of C. asiatica.

Micropropagation of orchids are often through the production of Protocorm Like Bodies (PLBs) and this research conducted a study to discover the optimal condition for the growth of Protocorm Like Bodies (PLBs) and also plantlets regeneration of Grammatophyllum speciosum Several aspects such as different carbon sources (sucrose, maltose) and the presence of charcoal alongside with the variety of concentration of agar were taken into account for the optimization. This study discovered that the 30 g/L of sucrose in 1 mg/L of BAP was superior in producing heavy PLBs. However, the trend differs in the number of plantlets where maltose (5 g/L) is seen to produce a higher number of plantlets compared to sucrose. Charcoal plays an important role in the growth of PLBs where the heaviest PLBs were recorded in the medium supplemented with 0.20 % of charcoal along with 4.5 g of agar. Vice versa relationship was recorded for the number of plantlets where without charcoal in 4.5 g of agar produce the highest number of plantlets.

Ninebark (Physocarpus opulifolius) is an attractive ornamental shrub with poor rooting characteristics in some cultivars, which is a limiting factor in commercial production This study was designed to optimize rooting conditions of ninebark cuttings and to observe the effect of exogenous auxin IBA on some morpho-anatomical and biochemical changes associated with rhizogenesis in the in vitro conditions. Both auxins under study: the indole-3-butyric acid (IBA) and 1-naphthalene acetic acid (NAA) gave comparable effects but the combination of ½ MS + 1 mg·L−1 IBA was the most cost effective for all rooting parameters. Anatomical changes at the cuttings’ bases during root formation were typical for woody plants and they were accelerated by auxin in the culture medium. High levels of the endogenous indole acid and hydrogen peroxide were temporarily associated with intensive cell divisions in cuttings, and the polyphenolic acid contents kept increasing during rooting above the initial levels and those in controls. Results of this work show that micropropagation may be the answer to growing demand for ninebark stocks. Anatomical changes occurring at the bases of stem cuttings during root formation in vitro are typical for woody plants. Auxin in the rooting medium accelerated individual phases of rhizogenesis making it shorter and more efficient. High levels of the endogenous indole acid and hydrogen peroxide were transiently associated with intensive cell divisions in cuttings. In auxin-treated cuttings, the polyphenolic acid contents increased during rooting above the initial levels and those in non-treated cuttings.

Paphiopedilum x dalatense is a beautiful orchid species with large flowers in variable colors and leaves covered with stripes and beautiful unseen mosaic spots. Recently, many people exploit this species, causing it becomes very rare. In this study, we studied the effects of various organic matter: potato, banana and tryptone, yeast powder, peptone on the growth and development of P. dalatense shoots as well as the effects of NAA and humic acid on in vitro rooting of this orchid were investigated. The research results showed that MS medium supplemented with 100 g/L banana in combination with 100 g/L potato (5,4 shoots/sample, 18,8 mm/shoot, 4,5 leaves/shoot, and shoots survival rate of 100%) or MS medium supplemented with 1 g/L peptone (4,19 shoots/sample, 15 mm/shoot, 4 leaves/bud, and 92% of shoots survival rate) were the best response for the shoot formation and development. In addition, the half strength MS culture medium supplemented with 1 mg/L NAA (5,2 leaves/sample, 4,6 roots/buds, 3,56 cm/root, and 100% rate for rooting) was the suitable medium for the in vitro rooting of P. dalatense. Being cultured on half strength MS medium supplemented with 2 mg/L humic acid, the rooting rate reached 100% with the greatest root number and the longest root (5 roots/shoots, 5,5 cm/root). The obtained results on the in vitro propagation on this orchid helps contribute to the conservation and increases the genotic pool of this precious wild orchid species, as well as the rapid multiplication of healthy plantlets serving the commercialization of precious orchid species.

Micropropagation was successfully established in the pineapple orchid Dendrobium densiflorum for its conservation and future utilization in the anticancer drug discovery. Micropropagation was carried out via seeds culture on the MS medium. Furthermore, antioxidant and anticancer activities were explored through DPPH and MTT assays. For culture initiation, maximum seed germination (84%) was achieved on half-strength MS medium supplemented with 10% coconut water. Full-strength MS medium supplemented with 15% coconut water was found suitable for highest number of shoot formation from protocorms, while a maximum number of roots were developed on micro-shoots grown on the full-strength MS medium supplemented with 1.5 mg/L IBA. The combination of cocopeat, pine bark and sphagnum moss in the ratio of 2:1:1 was the best substrate used in acclimatization stage resulting in 92% survivability of acclimatized plantlets. Extract of wild plant’s stems (DDW) at 475.28 μg/mL concentration inhibited the 50% DPPH free radicals. Therefore, DDW at 369.16 and 470.97 µg/mL concentrations inhibited the growth of 50% cervical cancer cells (HeLa) and glioblastoma cells (U251) respectively. This research highlighted the micropropagation of D. densiflorum to be utilized in conservation and its potential anticancer drugs to be discovered in future.

Bamboos (Bambusoideae, Poaceae) are multiple-purpose perennial grasses, which display a growing production chain in Brazil. One of the main bottlenecks is high-quality supplying of plantlets, then requiring efficient mass propagation methods, such as micropropagation. Contamination by microorganisms is recurrent in bamboo in vitro cultures, although some of those manifestations are considered endophytes harboring plant growth promotion potential. The isolation of endophytic bacteria from in vitro cultures of Dendrocalamus asper and Bambusa oldhamii was performed to assess their potential growth-promoting effect in co-cultivation with in vitro plants of Guadua chacoensis, an economically promising bamboo species. Among the total bacterial collection (32 isolates), all of them showed growth-promotion potential as indole compounds-producers. Sequences of 16S rRNA genes from eight selected isolates were newly generated, and the BLASTn similarity test recovered four bacterial genera (Bacillus, Brevibacillus, Serratia, and Atlantibacter) and six species. The co-cultivation experiment was carried out with three isolates selected based on their low- (Ba16), medium- (Ba03), and high-yield (Ba24) production of indole compounds, and Bayesian inferences strongly supported them as Bacillus subtilis, Serratia marcescens, and Brevibacillus parabrevis, respectively. The co-cultivation with three bacterial isolates, and their dilution levels, did not influence shoot or root growth and, however, did not cause apparent impairment for G. chacoensis in vitro cultures. Taken together, the isolation of endophytic microorganisms from field-growth bamboo clump and its co-cultivation with in vitro cultures of bamboos is possible, encouraging a continuous discovery and improvement of micropropagation techniques.

lbgBackground and Objective:l/bg The cat whiskers plant (ligOrthosiphon aristatusl/ig Blume Miq) is a plant that has been widely used as raw material for traditional medicine. The population of white-purple varieties of ligO. aristatusl/ig is decreasing efforts to maintain the white-purple ligO. aristatusl/ig need to be done keeping in mind its potential as raw material for traditional medicine. This study aims to determine the composition of a suitable medium in growing plantlet ligO. aristatusl/ig white-purple varieties and the content of its secondary metabolites. lbgMaterials and Methods:l/bg The internode explants were induced on MS medium added by various combinations of zeatin and 2,4-Dichlorophenoxyacetic acid (2,4-D). Root induction was carried out on shoots formed on MS medium with Indole-3-Butyric Acid (IBA). The acclimatization process was carried out using soil media. Determination of secondary metabolite levels was carried out on ligO. aristatusl/ig (ligin vitrol/ig culture) and wild-type plants aged ten months using high-performance liquid chromatography (HPLC). lbgResults:l/bg MS+BAP 2ppm+NAA3 ppm media was the optimal medium for growing shoots in leaf explants. Media MS+zeatin 3 ppm+2,4-D 2 ppm produced good shoot growth on internode explants. The best root induction occurred in MS+IBA media of 0.75 ppm. The acclimatization process was successful on shoots originating from the internode, while those from leaf explants had not succeeded in growing and developing. lbgConclusion:l/bg The levels of rosmarinic acid and sinensetin in the white-purple variety ligO. aristatusl/ig (ligin vitrol/ig culture) were 1.08 and 1.62% w/w and higher than those of wild varieties.

Demand for therapeutic honey is driving establishment of Leptospermum plantations. This study developed micropropagation methods for two species – Leptospermum polygalifolium Salisb. and L. scoparium J.R.Forst. & G.Forst. The study determined how shoot proliferation and adventitious rooting were influenced by the original explant position on the seedling and the concentration of benzyladenine (BA) in the proliferation medium. Hormone-free node culture was highly effective for both species. Nodal explants often formed roots in the absence of BA and developed elongated axillary shoots. Median shoot numbers of 584 and 659 were formed in 31–32 weeks from a single L. polygalifolium or L. scoparium seed, respectively. A low BA dose was effective for callogenesis and shoot proliferation of L. polygalifolium, but not L. scoparium. The median number of shoots produced from a single L. polygalifolium seed was 630 using 2.22-μM BA. This dose induced extremely high shoot numbers in some clones because explants often produced extensive callus and multiple short shoots. Shoots formed adventitious roots without indole-3-butyric acid and plantlets were acclimatised to nursery conditions. The original explant position did not influence shoot proliferation or adventitious rooting. Leptospermum polygalifolium and L. scoparium proved amenable to micropropagation, facilitating rapid establishment of nectar plantations.

Lippia graveolens u orégano mexicano, es una planta de importancia económica y la recolección del follaje se realiza principalmente de poblaciones naturales. La micropropagación propone una alternativa para la producción de material, con alta calidad fitosanitaria y propagación en menor tiempo de individuos seleccionados por sus características de valor comercial. El objetivo de este trabajo fue establecer un protocolo para la propagación in vitro de Lippia graveolens. La investigación se realizó en el año 2018-2019. El establecimiento se logró con segmentos nodales en medio basal MS, en esta etapa se recuperó 57.32% de explantes axénicos. Para la etapa de propagación se adicionó al medio MS cuatro concentraciones (0, 1, 2 y 3 mg L-1) de bencilaminopurina (6-BAP), se evaluó número de brotes y altura de plántula, los mejores resultados en la multiplicación se lograron con 3 mg L-1 de 6-BAP, obteniéndose 40.35 brotes por explante. La longitud de brote fue mayor en los tratamientos sin reguladores de crecimiento. Para el enraizamiento, se ensayó con tres concentraciones (0, 0.1, 0.3 y 0.5 mg L-1) de auxinas (ácido indol acético y ácido indol butírico) y se evaluó el porcentaje de enraizamiento y la longitud de la raíz obteniéndose igual resultado con y sin reguladores auxínicos. En la fase de aclimatación se evaluó el fotoperiodo (16 h y 24 h) con y sin micorrizas (Glomus intraradicens). El mejor tratamiento se obtuvo con 24 h luz y con adición de micorrizas con un 86.11% de supervivencia.

Carnation is an important cut flower in the floriculture industry. A micropropagation method for carnation cv. ‘Irene’ has been standardized. Nodal plant explants were grown in Murashige and Skoog (MS) medium containing plant growth regulators (PGRs). Determination of clonal fidelity of regenerated plantlets was performed with the help of SSR markers. Early formation of micro-shoots with maximum regeneration was noted in the half MS medium in combination of benzyl amino purine (BAP) thidiazuron (TDZ) and silver nitrate (AgNO3) in the concentrations 1.5, 0.25 and 0.5 mg L−1, respectively; while BAP (2.5 mgL−1), TDZ (1.0 mg L−1) and AgNO3 (1.5 mg L−1) produced maximum length of the micro-shoots. Quick induction of roots and maximum number of roots per culture were achieved in half MS basal rooting medium supplemented with 1.5 mg L−1 of indole-3-butyric acid (IBA) and 0.5 mg L−1 of naphthalene acetic acid (NAA). Simple sequence repeat (SSR) markers for clonal fidelity analysis showed uniform genetic fidelity among the proliferated plantlets and the mother plant lines. The protocol can be suitable for commercial cultivation of carnation plants.

Santalum album L. (Indian sandalwood) is an economically important but vulnerable tropical tree species. Cultures were established via direct shoot regeneration from axillary buds on Murashige and Skoog (MS) medium supplemented with 2.5 mg L−1 6-benzylaminopurine (BAP). The shoots were multiplied using MS medium containing 1.0 mg L−1 BAP and 0.5 mg L−1 indole-3 acetic acid and rooted on half strength MS medium containing 1.0 mg L−1 indole-3 butyric acid. The rooted plantlets were hardened and acclimatized in greenhouse using soilrite® and cocopeat (1:1) mixture. The concentrations of photosynthetic pigments were analyzed and detected less under in vitro conditions (6.05 μg g−1 FW) as compared to the 4 weeks old hardened (6.91 μg g−1 FW) and 12 weeks old acclimatized plantlets (7.8 μg g−1 FW) under greenhouse (ex vitro) environment. The anatomical evaluation of plantlets at subsequent stages of propagation suggested that the in vitro raised plantlets possessed structural abnormalities such as underdeveloped cuticle, unorganized tissue systems, reduced mesophyll tissues, fewer vascular elements and mechanical tissues, and loosely arranged thin walled paranchymatous ground tissues, which were slowly repaired during ex vitro hardening and acclimatization process to validate the developmental adaptation of micropropagated plantlets for maximum survival in the field (98.0% survival rate). The findings could help in the optimization of high-frequency commercial micropropagation of S. album for year-round production, and supply of this economically prominent vulnerable plant species to the farmers and the industries that rely on it. The anatomical and physiological features at subsequent stages in micropropagation of Santalum album elucidated the in vitro anomalies and ex vitro repair mechanism for improved survival in field.

Endophytic microorganisms is a major constrain to the establishment and growth of tissue culture plants. We report the use of copper sulfate (CuSO4.5H2O) to eliminate the endophytic bacteria in Philodendron selloum in vitro cultures. Contaminated shoots were cultured onto Murashige and Skoog (MS) medium containing 5 mg/L BA and supplemented with different concentrations of copper sulfate at 0, 35, 70 and 140 mg/L for 6 weeks. Copper sulfate at 70 mg/L completely eliminated the endogenous bacteria without decline in plant growth. However, 35 mg/L copper sulfate was optimal for maximum shoot multiplication (25), survival percentage (100%) and growth of plants. Antioxidant enzymes activity of catalase, peroxidases, and polyphenol oxidase were increased because of copper sulfate treatments. Conversely, electrolyte leakage was decreased at low copper sulfate (≤70 mg/L). Randomly amplified polymorphic DNA (RAPD) analysis revealed that plantlets exposed to different levels of copper sulfate were not genetically different from control plants.

A reliable and successful micropropagation protocol was developed for pepino plant (Solanum muricatum Aiton) from nodal segment explants grown on MS medium. The best values of shoot multiplication traits were recorded from the addition of 3 mg.l-1 kinetin by producing 2.3 shoots/explant, 3.6 cm and 9.6 leaves/ explant which was significantly superior upon the addition of BA at the same levels. In case of adding 3 mg.l-1 kinetin, the best root formation attributes were achieved from the use of 0.2 mg.l-l IAA that resulted a maximum number of roots (14.33 roots/ explant). The longest root length (15.33 cm) was achieved when 0.3 mg.l-l IAA was used. A 100% rooting percentage was recorded from the all tested auxins including IAA, IBA and NAA. The gradually moved plantlets from the heterotrophic phase in the lab to the autotrophic phase in the greenhouse showed 100% success. The plantlets did not show any abnormal growth or morphological changes. It is concluded that this important plant can be easily propagated by tissue culture technique through a reliable micropropagation protocol.

Micropropagation is a promising technique for hop (Humulus lupulus L.) plantlet production. However, the phenotypic stability of micropropagated hop plants is poorly understood. These should be characterized, in order to identify possible variations. The purpose of this study was to analyze and characterize the occurrence of somaclonal variation induced by in vitro multiplication of hop genotypes. Three genotypes were established in vitro and micropropagated in five subcultures. The response variables were chosen by the stepwise method and subjected to multivariate analysis of variance (Manova), multivariate contrasts, standardized canonical coefficients (StCC) and intraclass correlation (t). Somaclonal variation was detected in the genotypes Chinook and Columbus, both from America. No significant variations were found in the European genotype Hallertauer Mittelfrueh, regardless of the number of subcultures. Somaclonal variation was detected in the genotypes Chinook and Columbus, both from America. No significant variations were found in the genotype Hallertauer Mitterlfrueh, regardless of the number of subcultures.

Aloeswood or Agarwood has long been a perfumery commodity traded between Mediterranean Region and Southeast Asia since B. C. era. Oil or chips obtained by destructive harvesting of several Thymeleaceous genera including Aquilaria and Gonystylus are expensive and such ingredients are high in demand in the global market. The recent discovery of Gyrinops walla as a potential producer of market-quality agarwood in mature damaged woods and branches, intensified illicit felling and exportation of G. walla that led to it being in the verge of extinction from Sri Lankan flora. The sustainable utilization of G. walla undoubtedly enhances the foreign exchange of the country and the non-destructive utilization G. walla through tissue culture-based techniques is the only option available for sustainable exploitation and conservation of the vulnerable G. walla species. Micropropagation of G. walla was achieved by varying concentrations of BAP and NAA on MS medium to produce shoot and root with leaf-derived callus, respectively. The phytohormone concentrations of 3.0 mg/l BAP and 2.0 mg/l NAA proved the optimum concentrations for shoot and root induction, respectively. The callus turned to green and produced fewer buds. Roots were protruded out from the calli in the root induction medium. The findings of the study led to conclude that the micropropagation was viable in potential as an in vitro system for sustainable utilization and conservation endeavours of G. walla.

A study was conducted to standardize a protocol for in-vitro direct regeneration and mass multiplication of Citrus indica Tanaka using shoot tip explants excised from mature trees. Shoot tips were inoculated in MS medium supplemented with varying concentrations and combinations of cytokinins and auxins. MS media when fortified with BAP 0.5 mg/l and 0.5 mg/l BAP + 1.0 mg/l Kn were found to be the best treatments for shoot initiation while MS supplemented with 1 mg/l IBA; 0.5 mg/l NAA + 0.5 mg/l IBA and 0.5 mg/l NAA + 0.5 mg/l IAA were the best treatments for root induction. Among the different media used for hardening, 100 % survivability was obtained when plantlets were hardened using vermicompost as the potting medium. Subsequently, these plantlets were transferred to larger pots and acclimatization was achieved gradually in outdoor conditions. Plant Tissue Cult. & Biotech. 31(1): 13-23, 2021 (June)

A high frequency in vitro plant regeneration of pomegranate was established on MS medium supplemented with different concentrations and combinations of plant growth regulators. As explant cotyledons were employed for this study. Ninety percent of the cultured explants responded to form shoots from 30 days old in vitro raised seedlings after 90 days of culture initiation in MS containing 1.0 mg/l IBA + 0.1 mg/l NAA. The average number of shoots per explant was 10.0 ± 2.20, shoot length of 12.0 ± 2.40 cm, node per regenerated shoot was 9.0 ± 1.60 and the leaf number was14.0 ± 1.40. Well developed shoots were cultured on half strength of MS medium supplemented with 0.5 mg/l IBA, in which 90% shoot induced roots implanted after one month. The average number of root per shoot was 8.0 ± 0.90 and the average root length of 6.5 ± 0.40 cm was observed in this medium. Eighty percent plantlets were survived in the outdoor condition during the acclimatization period of seven days. Plant Tissue Cult. & Biotech. 31(1): 61-69, 2021 (June)

Within the nursery sector, a global interest in functional fruits imposes a permanent need to improve the tissue culture technology. A modern bioreactor immersion system is currently a valid solution for the micropropagation of many cultures. At the Faculty of Agrobiotechnical Sciences Osijek (FAZOS, Croatia), a research was conducted with the aim of improving the raspberry (Rubus idaeus L., cv. HimboTop®) micropropagation protocol. The treatments implied the modifications of the MS and DKW nutrient medium (DKW – standard; DKWm – substitution of FeEDTA with the FeEDDHA; MSm – macroelement modification, 3 x CaCl2, MgSO4, and KH2PO4 + FeEDDHA), differently depending on the aggregate condition (semi‐solid and liquid SETISTM). Subsequent to 15 (SETISTM) and 30 cultivation days (semi-solid medium), respectively, a morphological parameter analysis and a multiplication success evaluation were commenced. In all treatments (DKW, DKWm, and MSm), the observed parameters (shoot height, number of leaves, leaf width and length) were significantly better in an interaction with the liquid medium. Comparing the models, a liquid medium (SETISTM) resulted in a significantly higher explant multiplication (4.02) (a semi‐solid medium - 2.74). The best reproduction quality, implying a better plant vigor with a reduced vitrification, was obtained on the MSm medium in the SETISTM. A concentration optimization of individual mesocomponents (i.e., of the macroelements) and their chemical forms (FeEDDHA) may contribute to their improved adoption and consequently to the stress suppression due to explant transplantation. Selecting the modern technical solutions (TIS bioreactors - SETISTM) and continuously improving the protocols, we may obtain the realistically sustainable models, suitable for a massive clonal reproduction.

Resumen: El lapacho negro es una especie forestal de gran importancia, es maderable y ornamental, por lo que el establecimiento de un protocolo de micropropagación de Handroanthus heptaphyllus podría constituirse en una alternativa para la producción masiva de esta especie. La experimentación se llevó a cabo en el Laboratorio de Biotecnología del Área de Biología de la Facultad de Ciencias Agrarias de la Universidad Nacional de Asunción, teniendo como objetivo establecer un protocolo para la micropropagación in vitro de Handroanthus heptaphyllus a partir de segmentos nodales. Para la población de variables y unidades fueron sembrados 1620 explantes, cada unidad experimental estuvo constituida por 1 segmento nodal. La experimentación tuvo 12 tratamientos sembrados en dos medios de cultivo (Agar/Agua y MS), los cuales se basaron en variaciones en tiempos de inmersión de etanol e hipoclorito de sodio para el control de contaminación, para el control de oxidación se aplicó el tratamiento de prevención de oxidación 2 g/l de carbón activado, y fueron adicionados fitorreguladores (AIB y BAP). Las plantas madre fueron tratadas con Oxicloruro de cobre (OxiCob®) y Ditiocarbamato (Mancozeb®) en proporción de 3 y 2 g/l respectivamente. Las variables estudiadas fueron el control de manchas foliares en plantas madre, la contaminación de explantes, la oxidación, la sobrevivencia, la brotación, la formación de callos y la longitud de los brotes. El análisis estadístico consistió en pruebas de normalidad de Kolmogorov-Smirnov para todas las variables. Se logró un 45,8% de control de manchas foliares, un 50% de control de la contaminación y la oxidación fue controlada al 100%. Se obtuvo un 60% de explantes vivos hasta los 50 días con diferenciación foliar de 1,5 mm de elongación promedio. Con base en estos resultados se concluye que los tratamientos aplicados incidieron en las variables estudiadas. Abstract: The lapacho negro is a forest species of great importance, it is timber and ornamental, so the establishment of a protocol for micropropagation of Handroanthus heptaphyllus could become an alternative for the mass production of this species. The experimentation was carried out in the Biotechnology Laboratory of the Biology Area of the Facultad de Ciencias Agrarias de la Universidad Nacional de Asunción, with the general objective of establishing a protocol for the in vitro micropropagation of Handroanthus heptaphyllus from nodal segments. For the population of variables and units, 1620 explants were planted, each experimental unit consisted of 1 nodal segment. The experimentation had 12 treatments sown in two culture media (Agar/Water and MS), which were based on variations in immersion times of ethanol and sodium hypochlorite for contamination control, for oxidation control the treatment was applied of oxidation prevention 2 g/l of activated carbon, and phytoregulators (AIB and BAP) were added. The mother plants were treated with Copper Oxychloride (OxiCob®) and Dithiocarbamate (Mancozeb®) in a proportion of 3 and 2 g/l respectively. The variables studied were the control of leaf spots in mother plants, contamination of explants, oxidation, survival, sprouting, callus formation and the length of the shoots. Statistical analysis consisted of Kolmogorov-Smirnov normality tests for all variables. 45.8% leaf spot control was achieved, 50% contamination control, oxidation was 100% controlled. 60% of living explants were obtained up to 50 days with leaf differentiation of 1.5 mm of average elongation. Based on these results, it is concluded that the applied treatments affected the variables studied.

Glycyrrhiza glabra, member of Leguminosae family, is economically valuable due to its medicinal properties. Today it is considered as an endangered plant, therefore establishing a rapid and efficient system for micropropagation of this plant is desirable for their protection. The present study standardized the micropropagation protocol for G. glabra involving callus induction and shoots regeneration with explants viz. leaf and stem. Our finding suggests that the use of only one sterilant is not efficient for the sterilization of licorice explant. HgCl2 (0.1%) for 5 min and ethanol (70%) for 5 min was found best when used along with prior washing and surface sterilization with tween-20 and bavistin. Callus induction was achieved on MS medium containing various concentration of BAP, 2,4-D and NAA. Maximum callus induction was observed on MS medium containing 2 mg/l BAP, 0.5 mg/l 2,4-D and 50 mg/l ascorbic acid with both leaf and stem explants. Production of callus using leaf as an explant was better than that of stem. The development of shoots from callus was initiated on MS medium containing BAP and IAA. The most efficient shoot formation was observed with 4 mg/l BAP, 0.2 mg/l IAA and 60 mg/l ascorbic acid. Best rooting was recorded with 3 mg/l IBA. Histological aspect of indirect organogenesis of G. glabra were evaluated. Successful multiplication of shoots from explants leads to the production of improved quality and great quantity of planting material.

The aimof this study was to establish a low-cost alternative protocol for micropropagation ofSolanum lycopersicumL. var.cerasiforme,popularly known as cherry tomato. In the in vitroestablishment, culture mediacontaining Laboratory Reagent-grade (LR)and commercialsucrose and varied concentrations of corn starch and agarwere tested. The replacement of thermal sterilization, usingautoclave,withchemicalsterilization,adding sodium hypochlorite (2%) in the medium, was also evaluated. In the multiplication stage,the mediumwas supplemented with agar and/or corn starch and commercial sucrose.Forrooting, agrowth regulator-free medium withcommercialsucrose supplemented with agar and/or starch was used. Themicroplants were thentransplanted into plasticcontainerscontainingonlygarden substrateandsubsequentlyacclimatized in a greenhouse.The results make it possibleto conclude that the reduction of costs in the micropropagation of cherry tomatocan beobtained by replacing LRsucrose with commercial sucrose,and by the use of chemical sterilization of the culture medium withsodium hypochlorite. The replacement of agar withcorn starch can be done partially, in the stages of establishment and multiplication,and totally, during rooting