Search Results (8)

Sweet basil (Ocimum basilicum L.) is one of the most well-known aromatic herbs, which are economically important for food and pharmaceutical purposes. In vitro regeneration protocols are a fundamental part of molecular approaches, such as genome editing, which are used to enhance crop quality and pathogen resistance. In this research, in vitro regeneration methods were developed to examine the morphogenic aptitude of four different explant types from five commercial cvs of Ocimum basilicum L. (‘Prospera’, ‘Paoletto’, ‘Italiko FT’, ‘Dark opal’, and ‘Bolloso napoletano’). ‘Prospera’ showed the highest direct regeneration efficiency in all of the explant types (100% in the roots, 36% ± 0.02 in the cotyledons, 7.5% ± 0.2 in the hypocotyls, and 50% ± 0.04 in the cotyledonary nodes). The roots were found to be the most effective explant type, producing nodule-like meristems (100% in ‘Prospera’ and ‘FT Italiko’, 95.24% ± 0.01 in ‘Bolloso napoletano’), as precursors of shoots. Histological analysis was confirmed to be a suitable method to detect meristematic activity during the early morphogenic process and to evaluate the explants’ regeneration potential. Full article

Agrobacterium-mediated transformation systems using epicotyl explants have been widely used for genetic transformations of citrus. However, their application in lemons is severely constrained by browning of epicotyl tissues, which leads to an extremely low efficiency of transformation. In this study, we developed an optimized Agrobacterium-mediated transformation system using whole cotyledonary node explants of ‘Eureka’ lemon (Citrus limon), which significantly reduced tissue browning and enhanced transformation efficiency up to 14.48%. In addition, preparation of the whole cotyledonary node was simple and rapid, which reduced time and labor. This system facilitated efficient generation of transgenic lemon plantlets and provided a novel explant source for citrus transformation. Full article

This study evaluated the effects of N⁶-benzyladenine (BAP) seed preconditioning and seedling-derived explants on in vitro plant regeneration potential in soybeans (Glycine max [L.] Merrill.). The findings showed that seed preconditioning with 2.55 mg/L BAP prior to germination significantly influenced seedling establishment and the development of shoots, shoot elongation, and rooting on MS media supplemented with BAP and TDZ, compared to the negative (MS-NC) and positive (MS-NP) controls. The results also showed significant differences based on the genotypes, with Dundee recording 91.0% germination over a minimum of 5 days, compared to 74.2% with Peking, followed by 87.5% and 80.0% overall shoot induction frequency in these genotypes, respectively. Regenerated shoots were successfully elongated on MS medium supplemented with 0.5 mg/L BAP plus 0.6 mg/L GA₃ and rooted on hormone-free medium, for 3‒4 weeks, and then hardened in the acclimatization growth room under elevated light levels. Overall, this study revealed that BAP preconditioning of seeds enhances the frequency of bud initiation and shoot proliferation, mostly in whole-seedling and cotyledonary node explants subcultured on MS-E and MS-A media supplemented with BAP in combination with TDZ. Full article

To accelerate breeding of Pinus massoniana Lamb. resistance to pine wilt disease (PWD), caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, a protocol was established for the in vitro propagation of P. massoniana and the evaluation of resistance of regenerated microshoots from different clones to PWN. Axillary bud induction was achieved by culturing cotyledonary node explants from 3-week-old seedlings in Gupta and Durzan (DCR) medium that was supplemented with 4 mg L⁻¹ 6-benzyladenine (BA) and 0.2 mg L⁻¹ α-naphthaleneacetic acid (NAA). Explants with induced buds were transferred to DCR medium without a plant growth regulator to facilitate elongation. Stem segments from elongated shoots were used as propagules for further shoot multiplication. Six-month-old regenerated shoots that met the requirements for a nematode resistance test were inoculated with aseptic PWN (500 PWNs/shoot). The wilting rate varied between clones from 20% to 100%, 18 days after inoculation. Except for Clone 227, which showed the highest resistance with a wilting rate of 0%, other clones showed wilting to various degrees 30 d after inoculation. The number of nematodes that were recovered from Clone 227 was significantly lower than from other clones. This study promotes the resistance breeding of P. massoniana to pine wilt disease and provides an effective method to study the host/pathogen interaction between PWN and P. massoniana. Full article

Tomato (Lycopersicon esculentum Mill.) as the most economically important vegetable crop worldwide has been investigated intensively for the development of new and improved varieties. Most of these technologies require efficient protocols for in vitro regeneration and propagation of plant material. In the present study, an efficient and reproducible in vitro regeneration system for five Romanian tomato genotypes (cvs. ‘Capriciu’, ‘Darsirius’, ‘Kristin’, ‘Pontica’ and ‘Siriana’) has been established. The tomato genotypes were selected based on their horticultural and economically valuable traits. To study the in vitro morphogenic response, various explants, such as cotyledons, cotyledonary nodes, hypocotyls, leaf explants, internodes, stem nodes and apical buds have been selected. The highest efficiency in terms of direct shoot organogenesis was obtained in cv. ‘Capriciu’ (98% for apical buds and 94% for stem nodes) on culture media with zeatin and indole-3-butyric acid. One advantage of this regeneration procedure is beside its feasibility in handling, the high percentage of regenerated shoots and their rooting. The present protocol contributes to the existing information regarding the response of tomato cultivars to in vitro culture conditions. Full article

Sweet basil (Ocimum basilicum L.; Fam. Lamiaceae) is an annual herbaceous plant with a high economic value used in folk medicine, pharmacology, and food production. In Italy, most of the varieties are used to produce the famous “pesto” sauce; however, almost all of them are susceptible to basil downy mildew (BDM) disease, strongly decreasing the growth of the fresh leaves and the survival of the whole plant. Nowadays, CRISPR/Cas9 technology is recognized to be a prominent way to enhance basil genetic breeding. In this work, we present an optimized protocol for in vitro direct regeneration of an elite cultivar, which is the major limiting factor for the transformation of O. basilicum. Regeneration has been obtained from different explants (leaves, cotyledons, cotyledonary nodes); the highest frequency has been obtained from cotyledonary nodes of seedlings germinated on MS medium containing TDZ. This protocol may be used for biotechnological applications as genome editing techniques to obtain basil-downy-mildew-disease-resistant clones. Full article

Cerastium candidissimum Correns is an endangered species. Nevertheless, its ornamental characteristics make it an ideal candidate for commercial use as a new ornamental and landscape plant. As there are no references to the in vitro propagation of this species, the present research aims to comparatively investigate the potential that in vitro propagation may have of three different populations of the plant, from Mounts Parnassos (PS), Parnitha (PR), and Hymettus (HM), respectively. The establishment of the initial cultures took place by cotyledonary- and stem-node explants, excised from in vitro grown seedlings, on Murashige and Skoog (MS) media, without hormone (Hf), or with 0.5 mg L⁻¹ 6-benzyladenine (BA). The shooting percentage was higher in the case of the PS population (97.6%). It was also higher for stem-node explants (93.9%). The multiplication phase took place on Hf MS, or on MS containing BA (0.5 and 1.0 mg L⁻¹), with or without 0.1 mg L⁻¹ 1-Naphthaleneacetic acid. The higher shoot production was observed in the PS and PR populations (88.0% and 76.9% shooting, with 3.1 and 2.7 shoots/explant, respectively). Micro-shoots rooted at high percentages on half-strength MS (80.0–100.0%). Successful acclimatisation (95.0%) was established on peat: perlite (1:1 v/v). Moreover, an efficient alginate-encapsulation procedure was developed from PS stem-node explants, using a 3.0% w/v sodium alginate and 14.7 g L⁻¹ calcium chloride solution. Alginate beads exhibited a higher shoot proliferation (3.8 shoots) after 60 days of storage at 4.0 °C and a 50-day growth on Hf MS than non-encapsulated in vitro node-explants did. The presented protocol of in vitro propagation of C. candidissimum could facilitate the exploitation of the species by the floricultural industry and contribute to conservation strategies. Full article

The tissue culture regeneration system of Lupinus albus has always been considered as recalcitrant material due to its genotype-dependent response and low regeneration efficiency that hamper the use of genetic engineering. Establishment of repeatable plant regeneration protocol is a prerequisite tool for successful application of genetic engineering. This aim of this study was to develop standardized, efficient protocol for successful shoot induction from cotyledonary node of white lupin. In this study, 5 day old aseptically cultured seedlings were used to prepare three explants (half cotyledonary node, HCN; whole cotyledonary node, WCN; and traditional cotyledonary node, TCN), cultured on four concentrations of M519 medium (M519, ½ M519, ^1/3 M519, and ¼ M519), containing four carbohydrate sources (sucrose, fructose, maltose, and glucose), and stimulated with various combinations of KT (kinetin), and NAA (naphthalene acetic acid) for direct shoot regeneration. High frequency of 80% shoot regeneration was obtained on ½ M519 medium (KT 4.0 mg L⁻¹ + NAA 0.1 mg L⁻¹) by using HCN as an explant. Interestingly, combinations of (KT 4.0 mg L⁻¹ + NAA 0.1 mg L⁻¹ + BAP 1.67 mg L⁻¹), and (KT 2.0 mg L⁻¹ + NAA 0.1 mg L⁻¹) showed similar shoot regeneration frequency of 60%. Augmentation of 0.25 g L⁻¹ activated charcoal (AC) not only reduced browning effect but also improved shoot elongation. Among the all carbohydrate sources, sucrose showed the highest regeneration frequency with HCN. Additionally, 80% rooting frequency was recorded on ½ M519 containing IAA 1.0 mg L⁻¹ + KT 0.1 mg L⁻¹ (indole acetic acid) after 28 days of culturing. The present study describes establishment of an efficient and successful protocol for direct plant regeneration of white lupin from different cotyledonary nodes. Full article