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De novo root regeneration is the process by which adventitious roots form around the wound site from wounded or detached plant organs. The de novo root regeneration process has been widely exploited in cutting technology used for vegetative propagation. Here, we employed detached leaf explants from Arabidopsis thaliana to form adventitious roots for studying the process of de novo root regeneration. GUS staining showed that the expression of CLAVATA3/EMBRYO SURROUNDING REGION-RELATED26(CLE26) was gradually increased surrounding the wound site of leaf explants during adventitious root formation. Semi-thin sections further showed that the expression pattern of CLE26 was closely linked to the formation of adventitious roots. Next, genetic analyses confirmed that the CLE26 gene was involved in de novo root regeneration. Furthermore, RNA sequencing (RNA-seq) of the leaf explants revealed that stress-related genes might be involved in CLE26-mediated adventitious root formation. Specifically, genes associated with the hydrogen peroxide catabolic process and oxidative stress response were predominantly upregulated in the cle26 mutant. In contrast, genes involved in the response to salicylic acid were largely downregulated in the cle26 mutant. Overall, our study indicates that the mutation in CLE26 might upregulate the expression of genes involved in reactive oxygen species metabolism or suppress the expression of genes associated with salicylic acid synthesis, thus promoting the formation of adventitious roots. These findings suggest that CLE26 is a potential candidate for the genetic improvement of adventitious rooting in cuttings. [ABSTRACT FROM AUTHOR]

Tetrastigma hemsleyanum is a perennial evergreen vine of the Vitaceae. The entire herb is used in traditional Chinese medicine as a broad-spectrum plant-based antibiotic, so it has high economic and social value. Wild T. hemsleyanum resources are scarce, so it has been declared an endangered and rare medicinal plant. Seed yield is low and vegetative propagation by cuttings results in limited plant production, so development of the T. hemsleyanum industry requires optimized propagation protocols and the development of new biotechnologies to proliferate this plant in commercial quantities. In this study, shoot organogenesis was successfully induced from leaves and petioles. Two plant growth regulators, 6-benzyladenine (BA) and thidiazuron, induced callus and adventitious shoots, but the ideal adventitious shoot induction medium was Murashige and Skoog (MS) medium containing 1.0 mg L−1 BA and 0.1 mg L−1 α-naphthaleneacetic acid (NAA). This resulted in a shoot proliferation coefficient (SPC) of 6.73 within 30 d at a light intensity of 100 µmol m−2 s−1. When light intensity was increased from 50 to 200 µmol m−2 s−1, SPC (7.35), chlorophyll a (Chl a), Chl b, and total Chl (a + b) content increased. On MS medium containing 0.1–2.0 mg L−1 NAA or indole-3-butyric acid, 100% of adventitious shoots formed adventitious roots. Plantlets showed no obvious morphological variation, and their survival exceeded 98% on a substrate of peat and river sand (v:v = 2:1). This study's protocols allow for the mass production of adventitious shoots for conservation purposes, and potentially for the commercial propagation of T. hemsleyanum. [ABSTRACT FROM AUTHOR]

Hieracium lucidum subsp. lucidum is a critically endangered endemic taxa of the Sicilian flora. It is a relict of the Tertiary period surviving on the cliffs of Monte Gallo (NW-Sicily). This research focused on finding the best protocols for seed germination and vegetative and in vitro propagation to contribute to ex situ conservation. Seed germination tests were carried out using constant temperatures of 15 ℃, 20 ℃ and 25 ℃ in continuous darkness and an alternating temperature of 30/15 ℃ (16 h/8 h, light/dark). The seeds had no dormancy, and a high germination capacity (70-95%) was obtained at all tested thermoperiods. The possibility of vegetative propagation of the taxon was evaluated through the rooting capacity of stem cuttings treated or not treated with indole-3-butyric acid (IBA). All cuttings were treated with IBA rooted within 2 months, while only 50% of the untreated cuttings were rooted within a longer time. An efficient protocol for rapid in vitro propagation from leaf portions was developed. The response of explants was tested on hormone-free Murashige and Skoog (MS) basal medium and MS enriched with different types of cytokinins: 6-Benzylaminopurine (BAP) and meta-Topolin (mT) in combination with naphthaleneacetic acid (NAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D) at the same concentration. The combination of mT (2 mg L-1) and 2,4-D (1 mg L-1) in the medium was the most effective and showed the highest percentage of callus induction and the mean number of regenerated shoots. The maximum rate of root regeneration and the maximum number and length of roots were obtained on hormone-free MS and MS enriched with IBA at concentrations of 1 mg L-1. From the results obtained, it can be concluded that H. lucidum subsp. lucidum can be successfully propagated using one of the tested techniques, subject to the availability of the material for reproduction. [ABSTRACT FROM AUTHOR]

Hieracium lucidum subsp. lucidum is a critically endangered endemic taxa of the Sicilian flora. It is a relict of the Tertiary period surviving on the cliffs of Monte Gallo (NW-Sicily). This research focused on finding the best protocols for seed germination and vegetative and in vitro propagation to contribute to ex situ conservation. Seed germination tests were carried out using constant temperatures of 15 °C, 20 °C and 25 °C in continuous darkness and an alternating temperature of 30/15 °C (16 h/8 h, light/dark). The seeds had no dormancy, and a high germination capacity (70–95%) was obtained at all tested thermoperiods. The possibility of vegetative propagation of the taxon was evaluated through the rooting capacity of stem cuttings treated or not treated with indole-3-butyric acid (IBA). All cuttings were treated with IBA rooted within 2 months, while only 50% of the untreated cuttings were rooted within a longer time. An efficient protocol for rapid in vitro propagation from leaf portions was developed. The response of explants was tested on hormone-free Murashige and Skoog (MS) basal medium and MS enriched with different types of cytokinins: 6-Benzylaminopurine (BAP) and meta-Topolin (mT) in combination with naphthaleneacetic acid (NAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D) at the same concentration. The combination of mT (2 mg L−1) and 2,4-D (1 mg L−1) in the medium was the most effective and showed the highest percentage of callus induction and the mean number of regenerated shoots. The maximum rate of root regeneration and the maximum number and length of roots were obtained on hormone-free MS and MS enriched with IBA at concentrations of 1 mg L−1. From the results obtained, it can be concluded that H. lucidum subsp. lucidum can be successfully propagated using one of the tested techniques, subject to the availability of the material for reproduction.

Nematanthus wettsteinii (Fritsch) H. E. Moore is one of the most popular ornamental species in China, and is known for its peculiar "goldfish" shape flower and is usually propagated by cuttings. This study aimed to establish in vitro protocols for N. wettsteinii regeneration. In this study, leaves and shoot-tips were used as explants, and the effects of adventitious shoot induction, rooting medium, and plant growth regulators (PGRs) on regeneration from explants were evaluated. Murashige and Skoog (MS) medium was used as the basic substrate. Among the PGRs tested, when leaves were used as explants, 0.1 mg L−1 α-naphthaleneacetic acid (NAA) and 2.0 mg L−1 thidiazuron (TDZ) induced the largest number of adventitious shoots (22.5 per leaf) with a 93.5% induction rate. When shoot-tips were used as explants, 3.0 mg L−1 benzyl adenine (BA) induced the fewest adventitious shoots (2.6 per shoot-tip), while 0.1 mg L−1 NAA and 2.0 mg L−1 TDZ yielded the largest number of adventitious shoot (20.3 per shoot-tip). The adventitious shoots were then transferred to MS medium containing NAA, BA, and gibberellin A3 (GA3) for shoot proliferation and elongation. When half-strength MS medium was supplemented with 0.3 mg L−1 indole-3-butyric acid (IBA), shoot rooting was essentially 100%. After transplanting the plantlets to garden soil mixed with river sand and vermiculite (1:1:1, v/v) substrate, 100% of the plantlets survived after 3 wk. These reliable regeneration protocols constitute a significant advance towards the large-scale propagation and conservation of this famous horticultural species. [ABSTRACT FROM AUTHOR]

Two models of Arabidopsis thaliana rhizogenesis in vitro were studied: (1) from the callus and (2) leaf explants petioles on the Murashige and Skoog one-tenth strength hormone-free nutrient medium and also with the addition of a growth regulator, such as indole-3-butyric acid. Morphological and anatomical studies show significant changes in the structure of the roots formed de novo in vitro from callus tissue, while the organs from leaf explant petioles were similar to those formed from seeds (primary). By the Sabinin–Kolosov method, a decrease in the percentage of the active root surface was established. The occurrence of structural changes during in vitro rhizogenesis and their effect on root functionality are discussed. [ABSTRACT FROM AUTHOR]

Wild plant species are of great interest as a source of pharmacologically valuable compounds but a great number of them are endemic and/or endangered ones. Modern plant biotechnology can provide reliable methods for their utilization without disturbing natural populations. In vitro culture methods for Rhodiola species are being intensively developed to include them into various biotechnological programmes. Aim. Development of a protocol for direct Rhodiola rosea L. plant regeneration from leaf explants. Methods. The leaves of R. rosea aseptically growing plants were used as the explants. Several variants of Murashige and Skoog (1962) agar-solidified culture medium supplemented with different combinations of auxins (1-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D)) and cytokinins (kinetin and 6-benzylaminopurine (BAP)) were estimated as potential regeneration-inducing media. Regeneration frequency was calculated as the percentage of leaves that produced shoots. Results. The use of MS medium supplemented with 2.5 mg/l BAP and 1.0 mg/l 2,4-D allowed inducing shoot formation with 100% frequency. An increase in the 2,4-D content up to 2.5 mg/l and decrease in BAP content to 1.0 mg/l resulted in decreasing of the regeneration frequency to 62.5%. Regeneration frequency was 25% and 62%, respectively, on the media containing 1.0 mg/l kinetin + 2.5 mg/l 2,4-D and 2.5 mg/l kinetin + 1.0 mg/l 2,4-D. Conclusions. R. rosea leaf explants have demonstrated high regeneration capacity with using the studied combinations of plant growth regulators. MS medium supplemented with 2.5 mg/l BAP and 1.0 mg/l 2,4-D allowed inducing shoot regeneration in leaf explants with the frequency of 100%. The frequency of regeneration was lower in the case of substitution of BAP for kinetin. The other types of morphogenesis (formation of adventitious roots and/or callus) were also observed.

Bitter melon (Momordica charantia L.), a widely cultivated food and medicinal plant native to the world's subtropics and tropics, is a Cucurbitaceae rich in carotenoids. However, the low seed germination frequency and progeny variability associated with the production of this plant have a substantial impact on its growth and yield. These constraints affect the availability and exploitation of this crop, especially the fruits, which are rich in secondary metabolites such as β-carotene and α-carotene. In vitro regeneration would help overcome the obstacle linked to the germination of this plant and increase its yield and utilization. A reproducible in vitro organogenesis protocol was established using bitter melon embryogenic callus derived from immature leaf explants of in vivo grown seedlings and in vitro plantlets. Regeneration via callus was conducted on MSB5 media augmented with different plant growth regulator concentrations. The maximum frequency of callus formation (95.09 %) was produced in MSB5 media incorporated with 1.2 mg L−1 NAA augmented with 0.5 mg L−1 TDZ. MSB5 medium with no growth regulators was observed to be the most suitable for the shoot and root formation from the callus, producing a significantly high shoot percentage of 90.91 % and 21.53 shoots per explants, and the highest rooting frequency and root number of 88.92 % and 6.23 roots per explant, respectively, from leaf-derived callus of in vitro plantlets. The elongated plantlets had grown to a significantly higher average height of 12.20 cm on media added with 0.75 mg L−1 GA3. This reproducible method for regenerating bitter melon plantlets could facilitate mass multiplication, conservation, and commercial field production.

This study was conducted to establish an efficient in vitro plantlet regeneration protocol using the ex vitro leaves as explants for Haworthia retusa. Leaf tissues were cultured on liquid full-strength Murashige and Skoog (MS) medium supplemented with 2.0 mg/L indole 3-butyric acids (IBA) for callus induction, followed by sub-cultured to solid medium for callus proliferation. Callus was then transferred to a fresh medium supplemented with 6-benzyl amino adenine (BA) for shoot development. The result showed that the maximum rate of shoot regeneration (100%), number of shoots per explant (43), and shoot height (9.4 mm) were recorded on the solid MS medium supplemented with 1.0 mg/L BA and 30 g/L sucrose. IBA improved rooting, whereas, NAA (naphthaleneacetic acid) causes calli to form at the base of the shoots. The half-strength MS medium supplemented with 0.5 mg/L IBA provided the best rooting response for the shoot. This medium formulation resulted in the highest rooting rate (100%) and the highest mean root number (5 roots/explant). The result of the present study would be helpful for the mass propagation of commercially important H. retusa. [ABSTRACT FROM AUTHOR]

This study was conducted to establish an efficient in vitro plantlet regeneration protocol using the ex vitro leaves as explants for Haworthia retusa. Leaf tissues were cultured on liquid full-strength Murashige and Skoog (MS) medium supplemented with 2.0 mg/L indole 3-butyric acids (IBA) for callus induction, followed by sub-cultured to solid medium for callus proliferation. Callus was then transferred to a fresh medium supplemented with 6-benzyl amino adenine (BA) for shoot development. The result showed that the maximum rate of shoot regeneration (100%), number of shoots per explant (43), and shoot height (9.4 mm) were recorded on the solid MS medium supplemented with 1.0 mg/L BA and 30 g/L sucrose. IBA improved rooting, whereas, NAA (naphthaleneacetic acid) causes calli to form at the base of the shoots. The half-strength MS medium supplemented with 0.5 mg/L IBA provided the best rooting response for the shoot. This medium formulation resulted in the highest rooting rate (100%) and the highest mean root number (5 roots/explant). The result of the present study would be helpful for the mass propagation of commercially important H. retusa.

This study was conducted to develop a protocol for in vitro shootmultiplication and callus induction of various mung bean varieties to obtain enhanced phytochemical content with the help of elicitors. For shoot multiplication, two types of explants (shoot tips and nodal tips) of three varieties of mung bean (Mung NCM-13, MgAT-7, and MgAT-4) were used. Both types of explants from in vitro and in vivo sources were cultured on the MS medium supplemented with different concentrations (0.25-3.0 mg/L, increment of 0.5 mg/L) and combinations of BAP and IBA as independent treatments. For callus induction, leaf explants (in vitro source) were cultured on MS medium supplemented with 2,4-D (1-3 mg/L) alone or in combination with BAP or NAA (0.5 and 1.0 mg/L). For the enhanced production of phenolics and glycosides, calli were cultured on MS media supplemented with zinc oxide (0.5 mg/L) and copper oxide nanoparticles (0.5 mg/L) as nano-elicitors. Results showed that in vitro explants responded better in terms of shoot length, number of shoots, and number of leaves per explant when compared to in vivo explants. Moreover, shoot tips were better than nodal explants to in vitro culturing parameters. All three varieties showed the optimized results in the MS medium supplemented with 1 mg/L BAP, while roots were produced only in cultures fortified with 1 mg/L IBA. The leaf explants of in vitro and soil-grown plantlets showed a maximum callogenic response of 90 and 80%, respectively, on MS medium supplemented with 2,4-D (3 mg/ml). Maximum phenolic content (101.4 µg of gallic acid equivalent/g) and glycoside content (34mg of amygdalin equivalent/g of plant material) was observed in the calli cultured on MS medium supplemented with 3 mg/L of 2,4-D. Furthermore, the addition of zinc oxide (0.5 mg/L) and copper oxide (0.5 mg/L) nanoparticles to the callus culture medium significantly enhanced the phenolic content of Mung NCM-13 (26%), MgAT-7 (25.6%), and MgAT-4 (22.7%). Glycosidic content was also found to be increased in Mung NCM-13 (50%), MgAT-7 (37.5%), and MgAT-4 (25%) varieties when compared to the control. It is suggested that elicitation of in vitro cultures of mung beans with nanoparticles could be an effective strategy for the enhanced production of secondary metabolites. [ABSTRACT FROM AUTHOR]

This study was conducted to develop a protocol for in vitro shoot multiplication and callus induction of various mung bean varieties to obtain enhanced phytochemical content with the help of elicitors. For shoot multiplication, two types of explants (shoot tips and nodal tips) of three varieties of mung bean (Mung NCM-13, MgAT-7, and MgAT-4) were used. Both types of explants from in vitro and in vivo sources were cultured on the MS medium supplemented with different concentrations (0.25–3.0 mg/L, increment of 0.5 mg/L) and combinations of BAP and IBA as independent treatments. For callus induction, leaf explants (in vitro source) were cultured on MS medium supplemented with 2,4-D (1–3 mg/L) alone or in combination with BAP or NAA (0.5 and 1.0 mg/L). For the enhanced production of phenolics and glycosides, calli were cultured on MS media supplemented with zinc oxide (0.5 mg/L) and copper oxide nanoparticles (0.5 mg/L) as nano-elicitors. Results showed that in vitro explants responded better in terms of shoot length, number of shoots, and number of leaves per explant when compared to in vivo explants. Moreover, shoot tips were better than nodal explants to in vitro culturing parameters. All three varieties showed the optimized results in the MS medium supplemented with 1 mg/L BAP, while roots were produced only in cultures fortified with 1 mg/L IBA. The leaf explants of in vitro and soil-grown plantlets showed a maximum callogenic response of 90 and 80%, respectively, on MS medium supplemented with 2,4-D (3 mg/ml). Maximum phenolic content (101.4 μg of gallic acid equivalent/g) and glycoside content (34 mg of amygdalin equivalent/g of plant material) was observed in the calli cultured on MS medium supplemented with 3 mg/L of 2,4-D. Furthermore, the addition of zinc oxide (0.5 mg/L) and copper oxide (0.5 mg/L) nanoparticles to the callus culture medium significantly enhanced the phenolic content of Mung NCM-13 (26%), MgAT-7 (25.6%), and MgAT-4 (22.7%). Glycosidic content was also found to be increased in Mung NCM-13 (50%), MgAT-7 (37.5%), and MgAT-4 (25%) varieties when compared to the control. It is suggested that elicitation of in vitro cultures of mung beans with nanoparticles could be an effective strategy for the enhanced production of secondary metabolites.

The aim of the present investigation was to optimize protocols for micropropagation and adventitious shoot regeneration from leaf explants of two wild ecotypes of red flesh apple Malus niedzwetzkyana for future breeding programs. At the proliferation stage, different concentrations of sodium nitroprusside (SNP) and triacontanol (TRIA) were compared. To optimize shoot regeneration from leaf explants, interactive effects of 1-phenyl-3-(1,2,3-thidiazol-5-yl)-urea – thidiazuron (TDZ), indole-3-butyric acid (IBA) and two explant types were investigated. At rooting stage, the effect of exposure time of microshoots to darkness and exposure time to different concentrations of IBA and α-naphthalene acetic acid (NAA) were compared. The results showed that SNP affected the growth rate significantly and the maximum multiplication rates per explant (9.6 in the first ecotype and 8.8 in the second) were produced in the Quoirin and Lepoivre medium containing 17 SNP µM, in addition to 4 µm 6-benzylaminopurine (BAP) and 3 µm gibberellic acid (GA3). IBA and TDZ affected the adventitious shoot regeneration from leaf explants significantly, the highest number of regenerated shoots (18.3 per explant) was obtained from the basal section of the leaves cultured on the medium containing 2 μM IBA and 15 μM TDZ. At rooting stage, the maximum rooting (88.6%) was obtained in the result of one weak exposure to darkness on medium containing 3 μM IBA.

An efficient method for direct somatic embryogenesis and callogenesis from date palm (Phoenix dactylifera L. cv. Estamaran) was established using leaf explants. The effect of pretreating leaf explants with three antioxidant combinations on the browning was studied. The results showed that both the pre-treatments were effective against the browning of explants. Surface sterilization of explants, without pre-treatment, enhanced the browning. After recognizing the importance of antioxidants, we cultured the explants on an MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1, 0.5, 1, 2, and 5 mg/l, thiadiazuron (TDZ) at 0 and 5 mg/l and 6-benzyladeninepurine (BAP) at 0, 5, and 10 mg/l. Callogenesis and direct embryogenesis on the surface of the leaf explants were observed. Only on the medium containing 5 mg/l 2,4-D, 10 mg/l BAP, and 5 mg/l TDZ globular embryos were formed directly on the leaf explants. The percentage of callus formation increased with an increase in the 2,4-D concentration; however, high concentrations of cytokinin caused a reduction of callogenesis. A medium supplemented with 5 mg/l 2,4-D, 5 mg/l TDZ, and 5 mg/l BAP, and 2 mg/l 2,4-D, and 5 mg/l BAP produced the highest number of calli per replication (2.7) on the leaf surfaces and leaf margins. Along with the advantages of direct somatic embryogenesis, this protocol facilitated the prospect of micropropagation without resorting to the intermediary callus stage, an important aspect to commercial production, and without sacrificing the plant by excising the shoot tips, which are commonly used for date palm tissue cultures.

Background: This research is intended to determine suitable types and concentrations of plant growth regulators (PGRs) to induce callus on stem and leaf sections of 4 species of the genus Garcinia, namely, Garcinia mangostana, Garcinia schomburgkiana, Garcinia cowa, and Garcinia celebica. The base mediumwasMSmediumcontaining 30 g l-1 sucrose, 0.5 g l-1 polyvinylpyrrolidone (PVP), and 7 g l-1 agar, and for the different treatments, PGRs were added to the medium as follows: thidiazuron (TDZ) at concentrations of 0, 0.1, 0.5, 1, and 2 mg l-1; 6-(3- hydroxybenzylamino) purine (meta-topolin) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; 4-amino-3,5,6- trichloro-2-pyridinecarboxylic acid (picloram) at concentrations of 0, 0.5, 2.5, and 5 mg l-1; and 2,4- dichlorophenoxyacetic acid (2,4-D) at concentrations of 0, 0.5, 1, 2, and 4 mg l-1. The occurrence of callus was observed after 4 weeks. Results: A maximum of 100% and 93% of G. mangostana leaf explants formed callus in the 0.5 mg l-1 and 1 mg l-1 TDZ treatments, respectively, while 100% of G. schomburgkiana stem explants formed callus in the 1 mg l-1 TDZ treatment and 89% of G. schomburgkiana leaf explants formed callus in the 0.5 mg l-1 picloram treatment. The highest callus induction rate for G. cowa was 62% in the 1 mg l-1 TDZ treatment and for G. celebica was 56% in the 0.5 mg l-1·mT-1 treatment. Conclusions: For all 4 species, the greatest amount of large nodular callus was observed in the TDZ treatments. White, friable callus was observed on most of the 2,4-D and picloram treatment groups. Most meta-topolin treatments resulted in minimal callus formation. [ABSTRACT FROM AUTHOR]

'Hansen 536' (Prunus dulcis 3 Prunus persica) is an important commercial rootstock for peach and almond. However, susceptibility to wet soil and bacterial canker has limited its use primarily to areas with less annual rainfall. Genetic engineering techniques offer an attractive approach to improve effectively the current problems with this cultivar. To develop an efficient shoot regeneration system from leaf explants, 10 culture media containing Murashige and Skoog (MS) or woody plant medium (WPM) supplemented with different plant growth regulators were evaluated, and adventitious shoot regeneration occurred at frequencies ranging from 0% to 36.1%. Optimal regeneration with a frequency of 32.3% to 36.1% occurred with WPM medium containing 8.88 mM 6-benzylamino-purine (BAP) and 0.98 to 3.94 mM indole-3-butyric acid (IBA). The regenerated shoots had a high rooting ability, and 80% of the in vitro shoots tested rooted and survived after being transplanted to substrate directly. Transient transformation showed an efficient delivery of the b-glucuronidase (GUS) reporter gene (gusA) using all three Agrobacterium tumefaciens strains tested with a concentration of OD600 0.5 to 1.0 for 4 days of cocultivation. The protocols described provide a foundation for further studies to improve shoot regeneration and stable transformation of the important peach and almond rootstock 'Hansen 536'. [ABSTRACT FROM AUTHOR]

An efficient in vitro plant regeneration method through somatic embryogenesis has been established in Haworthia retusa. Somatic embryos were induced from leaf explants on Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and α-naphthaleneacetic acid (NAA) either alone or in combination with 4 μM thidiazuron (TDZ). Of the four auxins studied, IBA was found to be the most promising in terms of somatic embryo induction, followed in decreasing frequency by 2,4-D, IAA, and NAA. The highest somatic embryo induction (60.7%), with a mean of 20.7 embryos per leaf explant, was observed on MS medium amended with 20 μM IBA. The inclusion of 4 μM TDZ to the auxin-containing medium significantly (p < 0.0001) increased the somatic embryo induction frequency as well as the number of somatic embryos. The best combination for somatic embryogenesis was IBA + TDZ. The highest incidence of somatic embryo induction (100%), with a mean of 55.8 somatic embryos, was obtained on a culture medium containing 16 μM IBA + 4 μM TDZ. Somatic embryos germinated best on MS medium supplemented with 2 μM gibberellic acid. Morphological variations were observed among the regenerated plantlets. Well-developed plantlets obtained from germination media were acclimatized in the greenhouse. [ABSTRACT FROM AUTHOR]

This study reports on in vitro regeneration of Abutilon indicum plantlets through callus mediated organogenesis. The leaf explants implanted on Murashige and Skoogs (MS) medium supplemented with 4.52 µM 2, 4-Dicholorophenoxy acetic acid (2,4-D) and 8.88 µM 6 Benzyladenine (BA) showed highest response (70.3%) for callus proliferation, but these callus did not showed any morphogenetic differentiation on the same medium even after 12 weeks. Whereas, subsequent sub-culture of this green proliferated callus on MS medium added with 2.68µM α-Napthalene acetic acid (NAA), 8.88µM BA and 543 µM Adenine sulphate showed the highest frequency (62.2%) of multiple shoot-buds production and also elongation of shoots. Well developed shoots were efficiently rooted in vitro on half strength MS medium supplemented with 7.38 µM Indole-3-butyric acid (IBA). Seventy per cent of in vitro regenerated plantlets were successfully established in garden and were morphologically alike to the donor plants. The genetic homogeneity of these in vitro regenerated plantlets was also affirmed by inter simple sequence repeat (ISSR) analysis using eight ISSR primers. This standardised in vitro organogenesis protocol supplements a good platform for the conservation of A. indicum germplasms and also caters for the needs of the herbal industry. [ABSTRACT FROM AUTHOR]

Water hyssop (Bacopa monnieri (L.) Pennell) is a medicinal plants. Its upper and lower halves of leaf explants were incubated in Murashige and Skoog (MS) medium supplemented with 0.25, 0.50 and 1.0 mg/L benzylaminopurine (BA) for 8 weeks; the explants were exposed to white (W) and red and blue (R and B, respectively) light-emitting diodes (LEDs), at 4:1, 3:1, 2:1 and 1:1 R and B light ratios, respectively. Shoot regeneration (100%) was achieved from all explants at all applied concentrations of BA and LED types. All explants showed different BA concentration requirements for regeneration of the maximum number of shoots. Longer shoots were obtained on medium with 0.25 mg/L BA. The W LED lighting system was found to be more effective for regenerating the maximum number of shoots (26.11) per explant (on the upper half of the leaf). Conversely, longer and shorter shoots were generated under 1:1 R:B and W LEDs, respectively. The number of shoots per explant ranged from 9.67-24.0 (full leaf), 6.33-25.92 (lower half of the leaf) and 7.33-27.33 (upper half of the leaf), respectively, in response to BA and LED light. Shoot length ranged from 0.94-1.90 cm (full lamina), 0.70-2.11 cm (lower half of the leaf) and 0.93-1.83 cm (upper half of the lamina) in response to BA and LED lifght. Regenerated shoots were successfully rooted using indole-3-butyric acid (IBA) and acclimatized in the aquarium provided with tap water.

Bitter melon ( Momordica charantia L.), a widely cultivated food and medicinal plant native to the world's subtropics and tropics, is a Cucurbitaceae rich in carotenoids. However, the low seed germination frequency and progeny variability associated with the production of this plant have a substantial impact on its growth and yield. These constraints affect the availability and exploitation of this crop, especially the fruits, which are rich in secondary metabolites such as β-carotene and α-carotene. In vitro regeneration would help overcome the obstacle linked to the germination of this plant and increase its yield and utilization. A reproducible in vitro organogenesis protocol was established using bitter melon embryogenic callus derived from immature leaf explants of in vivo grown seedlings and in vitro plantlets. Regeneration via callus was conducted on MSB5 media augmented with different plant growth regulator concentrations. The maximum frequency of callus formation (95.09 %) was produced in MSB5 media incorporated with 1.2 mg L -1 NAA augmented with 0.5 mg L -1 TDZ. MSB5 medium with no growth regulators was observed to be the most suitable for the shoot and root formation from the callus, producing a significantly high shoot percentage of 90.91 % and 21.53 shoots per explants, and the highest rooting frequency and root number of 88.92 % and 6.23 roots per explant, respectively, from leaf-derived callus of in vitro plantlets. The elongated plantlets had grown to a significantly higher average height of 12.20 cm on media added with 0.75 mg L -1 GA 3 . This reproducible method for regenerating bitter melon plantlets could facilitate mass multiplication, conservation, and commercial field production., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

An efficient method for direct somatic embryogenesis and callogenesis from date palm (Phoenix dactylifera L. cv. Estamaran) was established using leaf explants. The effect of pretreating leaf explants with three antioxidant combinations on the browning was studied. The results showed that both the pre-treatments were effective against the browning of explants. Surface sterilization of explants, without pre-treatment, enhanced the browning. After recognizing the importance of antioxidants, we cultured the explants on an MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.1, 0.5, 1, 2, and 5 mg/l, thiadiazuron (TDZ) at 0 and 5 mg/l and 6-benzyladeninepurine (BAP) at 0, 5, and 10 mg/l. Callogenesis and direct embryogenesis on the surface of the leaf explants were observed. Only on the medium containing 5 mg/l 2,4-D, 10 mg/l BAP, and 5 mg/l TDZ globular embryos were formed directly on the leaf explants. The percentage of callus formation increased with an increase in the 2,4-D concentration; however, high concentrations of cytokinin caused a reduction of callogenesis. A medium supplemented with 5 mg/l 2,4-D, 5 mg/l TDZ, and 5 mg/l BAP, and 2 mg/l 2,4-D, and 5 mg/l BAP produced the highest number of calli per replication (2.7) on the leaf surfaces and leaf margins. Along with the advantages of direct somatic embryogenesis, this protocol facilitated the prospect of micropropagation without resorting to the intermediary callus stage, an important aspect to commercial production, and without sacrificing the plant by excising the shoot tips, which are commonly used for date palm tissue cultures. [ABSTRACT FROM AUTHOR]

To preserve and propagate valuable Chinese cabbage (Brassica rapa L.) using mature vegetative organs without seeds, we established a high efficiency regeneration system from heading leaf explants and further explored its application in genetic transformation. Our results showed that maximum shoot regeneration of cultivars ‘Beijing New No. 3’ (88.46 ± 1.65%) and ‘Chengyangqing’ (73.77 ± 1.73%) were obtained in the presence of 17.76 μM 6-benzylaminopurine (BA) and 5.37 μM α-naphthylacetic acid (NAA). The addition of silver nitrate (AgNO3) at 11.77 μM was essential for shoot regeneration from heading leaf explants. Genotypic differences in shoot regeneration frequencies (from 47.24 ± 3.65 to 88.46 ± 1.65%) were observed amongst eight Chinese cabbage cultivars, in addition to the number of shoots per explant (from 1.31 ± 0.02 to 2.02 ± 0.05) in Murashige and Skoog medium containing 17.76 μM BA, 5.37 μM NAA and 11.77 μM AgNO3. Low temperature (4 °C) had an effect on in vitro preservation of leafy heads with a delay in leaf wilting, and there were no significant differences in shoot induction frequency within 24 h for cultivars ‘Beijing New No. 3’ and ‘Chengyangqing’. In the genetic transformation experiments using selection with kanamycin (17.17 μM), a transformation efficiency of 0.6-1.2% was achieved, as assessed from PCR and Southern blot results. The above results suggested that the heading leaf explants can not only achieve efficient seed-independent propagation of Chinese cabbage, but also provide a feasible platform for genetic transformation. [ABSTRACT FROM AUTHOR]

Incorporation of nanoparticles into a number of manufacturing products raised the concern of environmental release via deliberate or accidental routes. Here, experiments were performed to examine the effect of copper oxide nanoparticles (CuO NPs), and polyvinyl pyrrolidone (PVP) and polyethylene glycol (PEG) impregnated CuO NPs on seed germination and growth of Trigonella foenum‐graecum L. as well as on callus induction through tissue culture technique. Seed germination frequency, length, and weight parameters did not inhibit at higher extent by application of NPs; however, copper acetate, PVP, and PEG significantly decreased the values of all parameters. In all the cases, negative effects were observed concentration‐dependent. PVP and PEG impregnated CuO were found less toxic for calli fresh and dry weight induced from leaf and stem explants. The 2, 2‐diphenyl‐1‐picrylhydrazyl reagent‐free radical scavenging activity, total antioxidative potential, and total reducing power potential along with total flavonoid and phenolic contents are found elevated in root when compared with shoot. Furthermore, impregnation of PVP and PEG on CuO NPs increases the oxidative response. The results conclude that impregnation of organic molecules on nanoparticles does not reduce the toxicity though can be exploited for enhanced production of secondary metabolites for medicinal purposes. [ABSTRACT FROM AUTHOR]

Our experiments have confirmed the considerable disposition of leaf explants of Actinidia chinensis Planch. for induction and intensive proliferation of callus cultures, as well as, a possibility to regulate morhogenesis in in vitro conditions. Under specific culture conditions the morphogenic potential of callus cells of Actinidia chinensis was manifested both in organogenesis and somatic embryogenesis. Organogenesis was represented by induction of adventitious buds and regeneration shoots on the modified MS culture medium (Murashige and Skoog 1962) with BAP in combination with GA3 (each 1.0 mg. l-1). Rooting of shoots was successful on modified MS medium containing IBA (0.5-1.0 mg. l-1). Histological studies of callus tissues revealed their structural heterogeneity. Morphogenic processes in the callus were characterized by the appearance of meristematic zones and vascular elements. The formation of apical meristem, leaf primordia and finally shoot development proved de novo regeneration in callus culture. The obtained results demonstrate a possibility of plant regeneration through indirect organogenesis, which can be used for propagation of Actinidia chinensis Planch.

The developmental sequence of callus initiation and somatic embryogenesis in leaf explants of Cucumis sativus cv. Borszczagowski was analysed and compared on media containing two different auxin phenoxy-derivatives (2,4-D and 2,4,5-T) and cytokinin (BAP or 2iP). During the first 20 days of culture on media with 2,4,5-T proliferation of parenchymatic tissue occurred mainly and only small meristematic centers were observed. There was an intensive detachment of parenchymatic cells and dissociation of their cell walls near vessels and in the lower part of the explant adjacent to the medium. These cells were strongly plasmolysed. On the 2,4-D containing medium mostly meristematic tissue developed, proliferating around vascular bundles and forming meristematic centers or promeristem-like structures. After 35-50 days of culture, secondary callus was formed by separation of meristematic cells from the meristem surface in explants cultured on the 2,4-D containing medium. On medium supplemented with 2, 4, 5-T the detachment of parenchymatic and meristematic cells occurred, along with formation of a gel-like substance. The gel-like callus contained multi-cellular aggregates, proembryoids and embryoids. This type of callus tissue was initiated more intensively on medium with 2, 4, 5-T, but the frequency of somatic embryogenesis was much lower. The periferial cells of aggregates, proembryoids and embryoids showed the tendency to separate from the surface of the tissue. Many embryoids formed adventitious embryos.

Plant regeneration from C. anyuria var. longipes A. Meeuse and C. metuliferus Naud. leaf explants was investigated. It was found that embryoid-like structures were formed from C. anguria leaf explants cultured on media containing 0.3 mg dm-3 2,4-D and 0.8 mg dm-3 2iP. After being transferred to medium with 0.3 mg dm-3 2iP and 1 mg dm-3 GA3 they developed 187±49 shoots per explant. Further growth and root formation proceeded on hormone free medium.