Cryptomeria japonica (Thunb. ex L.f.) D. Don is a commercial tree native to Japan and is one of the most important forest species in that country and the Azores (Portugal). Because of the quality of ...C. japonica timber, several genetic improvement programs have been performed. Recently, some studies focusing on C. japonica somatic embryogenesis have been carried out. However, in this species, this process uses immature seeds as initial explants, and for this reason, it is not possible to achieve the maximum genetic gain (100% genetic of the donor plant). Although some studies have been made applying organogenesis to this species, the success of the process in adult trees is low. For this reason, our main goal was to optimize the micropropagation method by using trees older than 30 years as a source of plant material. In this sense, in a first experiment, we studied the effect of different types of initial explants and three basal culture media on shoot induction; then, two sucrose concentrations and two light treatments (LEDs versus fluorescent lights) were tested for the improvement of rooting. In a second experiment, the effects of different plant growth regulators (6-benzylaminopurine, meta-topolin, and thidiazuron) on shoot induction and the subsequent phases of the organogenesis process were analyzed. The cultures produced the highest number of shoots when QL medium (Quoirin and Lepoivre, 1977) and long basal explants (>1.5 cm) were used; the shoots obtained produced a higher number of roots when they were grown under red LED lights. Moreover, root induction was significantly higher in shoots previously induced with meta-topolin.
Persimmon (Diospyros) is an economically important tree widely cultivated for woody grain production in China, and Diospyros lotus is mainly used as the grafting stock of persimmon. However, the ...breeding of stress-resistant rootstocks of D. lotus using molecular means has yet to be achieved; in particular, an efficient blade-regeneration system has not been perfected to date. This study examined the effects of different plant-growth regulators and concentrations on the primary culture of stems with buds, the induction of leaf callus, the differentiation of adventitious shoots, and rooting culture of D. lotus. The optimal formula for inducing axillary buds from stems with buds was 1/2 Murashige and Skoog (MS) medium containing 2.0 mg/L 6-benzylaminopurine (6-BA) and 0.5 mg/L naphthaleneacetic acid (NAA), in which the induction rate of axillary buds approached 67.1%. The best medium formula for leaf callus induction was 1/2 MS medium containing 2.0 mg/L 6-BA and 0.5 mg/L NAA. Then callus was transferred to 1/2 MS medium containing 2.0 mg/L 6-(γ,γ-dimethylallylamino)purine (2iP), 2.0 mg/L thidizuron (TDZ), and 40 g/L sucrose to induce adventitious shoots after dark culture for 48 h, resulting in 7.9 shoots per explant and a 75.2% induction frequency of adventitious shoots. In addition, it was difficult to induce adventitious shoots from callus after six times of continuous transfer and differentiation. The adventitious shoots were transferred to 1/2 MS medium containing 2.0 mg/L zeatin (ZT) and 2.0 mg/L 2iP for proliferation culture, in which the multiplication coefficient approached 7.6. The adventitious shoots after multiplication were inoculated into 1/2 MS + 1.0 mg/L IBA + 0.5 mg/L NAA medium, the rooting rate was 70.2%, and the average number of heels was 9.6. Thus, studies in this area are expected to facilitate rapid and excellent growth, as well as theoretical support for factory saplings' care and molecular breeding.
An in vitro regeneration system for propagation of a valuable medicinal tree, Pterocarpus marsupium, using shoot tip (ST) expiants derived from 7-d-old axenic seedlings has been successfully ...developed. Murashige and Skoog (MS) medium containing cytokinins (BA, mT, or Kn) and auxins (NAA, IAA) in different concentrations and combinations showed to have a marked stimulatory effect on the regeneration output. Of cytokinins, meta-topolin (ml 7.0 μM) proved optimum dose for multiple shoot induction yielding 6.50 ± 0.49 shoots per expiant with mean shoot length (4.00 ±0.16 cm) in 70% cultures after 6 wk. Whereas, supplementation of low concentration of auxin (1.0 μM NAA) with optimal cytokinin (7.0 μM mT) favored enhanced shoot induction with increased percent response. At this level, a maximum of 13.54±0.34 shoots per expiant with (5.30±0.10 cm) mean shoot length were recorded in 80% cultures after 12 wk. Thereafter, microshoots were isolated and their basal end treated with high doses of indole-3-butyric acid (250 μM) for 5 d followed to transplant onto various potting substrates for ex vitro root formation. A maximum of 3.63±0.0.08 roots per microshoot and mean root length of 3.59±0.07 cm with highest rooting frequency (67.7%) was observed after 4 wk of transplantation. During ex vitro rooting-cww-acclimatization, various physiological parameters were found to be fluctuating initially for 35 d; thereafter, an increasing trend was observed in net photosynthetic rate, intercellular CO₂ concentration, and chlorophyll a/b estimation. Overall, the successfully acclimatized regenerants when transferred in natural condition showed about 96.7% survival rate after 3 mo. Assessment of genetic integrity of tissue culture raised plantlets was accomplished by DNA-based ISSR primers. A total of 35 bands with an average of 4.38 bands per primer were scored in monomorphic banding pattern. Thus, it is apparent that the mode of vitro regeneration protocol was suitable for obtaining true-to-type and disease-free plants.
Inula crithmoides L. (golden samphire) is an edible aromatic halophyte species with confirmed nutritional and medicinal properties attributed to the presence of important metabolites, including ...proteins, carotenoids, vitamins, and minerals. Therefore, this study aimed at establishing a micropropagation protocol for golden samphire that can serve as a nursery approach to its standardized commercial cultivation. For that purpose, a complete regeneration protocol was developed by improving shoot multiplication from nodal explants, rooting, and acclimatization methodologies. The treatment with BAP alone induced the maximum shoot formation (7–7.8 shoots/explant), while IAA treatment increased the shoot height (9.26–9.5 cm). Furthermore, the treatment that coupled best shoot multiplication (7.8 shoots/explant) and highest shoot height (7.58 cm) was MS medium supplemented with 0.25 mg/L BAP. Moreover, all shoots produced roots (100% rooting), and multiplication treatments did not exert significant effect on root length (7.8–9.7 cm/plantlet). Moreover, by the end of the rooting phase, plantlets cultivated with 0.25 mg/L BAP had the highest shoot number (4.2 shoots/plantlet), and plantlets from 0.6 mg/L IAA + 1 mg/L BAP presented the highest shoot height (14.2 cm) similar to control plantlets (14.0 cm). The survival up to the ex-vitro acclimatization stage was increased from 9.8% (control) to 83.3%, when plants were treated with a paraffin solution. Nevertheless, the in vitro multiplication of golden samphire is a promising way for its rapid propagation and can be used as a nursery method, contributing to the development of this species as an alternative food and medicinal crop.
An efficient and reproducible Agrobacterium tumefaciens-mediated transformation method was developed for Gossypium hirsutum L. 'KC3' using a shoot apex expiant with the combination of microinjection ...and sonication. To optimize the transformation efficiency, preculture conditions, acetosyringone concentration, A. tumefaciens cell density, and precision microinjection with sonication were evaluated. A high-throughput A. tumefaciens-mediated genetic transformation method was developed using the uidA gene. The shoot apex expiants were microinjected with an A. tumefaciens strain EHA105 carrying the uidA gene and cocultivated for 3 d on mMS medium fortified with 100 mM acetosyringone. Among the different parameters and conditions tested, preculturing expiants for 24 h, microinjecting three times with an A. tumefaciens cell suspension of OD600 nm = 0.6 containing 100 mM acetosyringone, and sonicating expiants for 20 s produced the maximum transformation efficiency of 20.25%. Putative transformants were transferred to regeneration medium supplemented with 0.8 mg L⁻¹ 1-naphthaleneacetic acid, 2 mg L⁻¹ thidiazuron, 16% (v/v) of an Oscillatoria acuminata extracellular extract, and 50 mg L⁻¹ kanamycin. Initially, the transgenic shoots were evaluated by GUS histochemical analysis, and integration was confirmed by polymerase chain reaction and Southern blot analysis. Plantlets transferred from an artificial growth chamber to greenhouse conditions showed a higher survival rate of 90% compared to the directly transferred plantlets. This simple and efficient transformation system could be valuable to transfer any gene of interest into cotton with high transformation efficiency in a short time.
Apennines genepi (Artemisia umbelliformis Lam. subsp. eriantha), is a high-altitude plant endangered by illegal and indiscriminate harvesting to produce the homonymous liqueur. Even if synthetic ...seeds represented a valid propagation technique to respond to this threat, this technology still requires methods to improve the low germination and plantlet development rates. The aim of the present study was to evaluate the suitability of the bacterial consortium formed by Azospirillum brasilense, Gluconacetobacter diazotrophicus, Burkholderia ambifaria, and Herbaspirillum seropedicae, as an enrichment agent of Apennines Genepi synthetic seeds. To evaluate the effectiveness as an improving agent, the consortium was incorporated in the synthetic seeds, compared to a control, and followed until ex vitro cultivations. Plants were evaluated for their growth and development parameters, total phenolic and flavonoid contents, antioxidant properties, polyphenolic compounds concentrations, and volatile fractions. The results demonstrated that the investigated consortium is a good candidate as an enrichment agent in synthetic seed technology. The rhizobacterial presence induced an improved response in plant propagation and the acclimatization process. Plant quality was also enhanced, with an increased accumulation of plant secondary metabolites and higher antioxidant capacity. The investigated bacterial consortium is a good candidate as an enrichment agent in synthetic seed technology. Plant clones can be translocated for species conservation and also commercialized to reduce the illegal and undiscerning collections.
Apios priceana, commonly known as Price's potato-bean, is a perennial species native to the Southeastern US. Habitat destruction has caused A. priceana to become rare, and it is federally listed as ...threatened. Protocols developed for in vitro germination, shoot micropropagation, in vitro rooting, shoot establishment in soil, and seed cryopreservation will assist in the safeguarding and conservation of dwindling natural populations. Seeds were germinated in vitro on plant growth regulator (PGR)-free Murashige and Skoog (MS) medium after seed sterilization in H₂O₂ and seed nicking. Greatest shoot multiplication occurred on MS medium with BAP/IBA/GA₃ at 2.22/0.49/1.44 µM with 2.0 g/1 Phytagel and pH adjusted to 5.7. Shoots rooted in vitro in MS medium with 3.2 µM IBA. Shoots rooted in vitro rapidly established in greenhouse potting mix, usually showing new growth within 2 wk, with tuber formation by the end of the growing season. Plants transferred to a forest setting in late winter survived, grew throughout the summer, and became dormant in the fall. In small post cryopreservation tests with A. americana and A. priceana seeds, air or desiccant-dried to water contents below 10% but above 2.5%, germination reached 87-90%.
Cicer microphyllum Benth. is a wild legume that is adapted to the extremely adverse climatic conditions of the cold Himalayan deserts. This rare species is a crop wild relative (CWR) that could be ...used for chickpea (Cicer arietinum L.) breeding programs to improve abiotic stress tolerance. However, the availability of C. microphyllum biological material is very limited due to its high altitude and remote location. These factors make in vitro plant tissue culture an excellent alternative for the conservation and propagation of this species. In this study, an efficient and reproducible in vitro regeneration protocol was established for C. microphyllum. This process enabled the regeneration of multiple shoots through indirect organogenesis from excised embryos. In addition, the effects of combinations of auxin and cytokinin concentrations in Murashige and Skoog (MS) medium were tested. The highest percentage of callus induction was observed on MS with a-Naphthaleneacetic acid (NAA) and 6-Benzylaminopurine (BAP), and the addition of AgNO₃ to MS with NAA and BAP significantly improved shoot morphogenesis. The highest root percentage was observed on half-strength MS with Indole-3-butyric acid Regenerated plantlets had a 65% survival rate 25 d after transplanting to garden soil, vermiculite, and vermicompost (1:1:1) potting mix. This protocol can be routinely used in large-scale propagation and possibly in germplasm conservation of this rare and important CWR species for additional use in chickpea breeding programs.
A new approach for rapid ex vitro rooting and acclimatization of Fragaria × ananassa micropropagated plantlets of two cultivars ("Alpha" and "Festivalnaya") has been developed using a ...mechanocomposite based on biogenic silica and green-tea catechins. Two different mechanocomposite treatments were studied: dipping the cut ends of microshoots in the mechanocomposite powder (the dry dip method) and single watering with solutions at concentrations of 0.3, 1.0, and 3.0 g L−1. These variants were compared with pulse treatment of microplants with 30 mg L−1 indole-3-acetic acid (IAA) for 4 h and a control group of microshoots that were moistened with hormone-free ¼-strength MS medium. The frequencies of ex vitro rooting at the end of the acclimatization period (30 d) varied from 24.8 to 99.7%. The dry dip treatment was best (rooting frequency about 100%) with up to 7.15 ± 0.54-cm root length, and 6.10 ± 0.31 roots per plantlet. Moreover, this study showed that the growth-stimulating effect of this mechanocomposite treatment on root formation resulted in increased rosette height, leaf number, leaf area, and dry weight of aerial parts. Histological analysis of the leaf blades revealed decreased mesophyll thickness of microshoots treated with the mechanocomposite (up to 88.77 ± 2.95 vs. 111.51 ± 3.56 μm for the control). Morphometric analysis of scanning electron microscopy data showed that mechanocomposite treatments led to increased stomata density and stomata length. These structural changes led to normalization of the water regime and indicated successful acclimatization. The combination of ex vitro rooting and acclimatization reduced the procedure time by 4 wk, and may be used for commercial strawberry micropropagation.
•The AFM technique is capable of distinguishing embryogenic calli from non-embryogenic.•Triclopyr outperforms picloram in the somatic embryogenesis of E. edulis.•Prematuration causes somatic embryos ...to multiply up to five times more.•The use of ABA in the maturation of E. edulis somatic embryos is efficient.
Euterpe edulis Martius is an endangered species of the Atlantic Forest, whose fruits have high antioxidant potential, and propagated exclusively by seeds. The present study assessed the ability of different auxin inducers and picloram analogs to trigger somatic embryogenesis in E. edulis. Immature seeds were harvested, and their zygotic embryos were excised and grown in MS culture medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram at 150, 300, 450, 600 µM. The activity of picloram analogs triclopyr and clopyralid was evaluated in semisolid MS medium. At maturation and germination, picloram-derived calli and somatic embryos isolated from triclopyr-grown cultures were first transferred to pre-maturation medium and, after 30 days, to basal MS or MS medium supplemented with either 5 µM abscisic acid or 0.53 µM 1-naphthaleneacetic acid plus 12.3 µM 2-isopentenyladenine. Finally, somatic embryos with root protrusions were transferred to MS medium devoid of sucrose for 30 days and then acclimatized ex vitro. Scanning, transmission, and atomic force microscopy revealed that picloram was superior to 2,4-D but less effective than triclopyr (100 µM) in inducing embryogenesis. Maturation and germination of somatic embryos in E. edulis can be maximized by 5 µM abscisic acid, and selecting calli via atomic force microscopy.
