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In vitro propagation of walnut has played a very important role in rapid multiplication of cultivars with desirable traits and production of healthy and disease-free plants. During the last several years, different approaches have been made for in vitro propagation of walnut. Micropropagation using apicale bud, nodale segement, leaves, petioles, co...
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... To date, extensive research has been conducted on the mechanism of oxidative browning of cultured tissues, including the identification of the browning-related genes and the modification of these genes in plant cells to reduce the oxidation of plant cells (Gao et al. 2020;Zhang et al. 2020). On the other hand, several attempts have been made to prevent the browning of tissue cultures by adding antioxidants or adsorbents to control the oxidation of phenolic compounds (Tang et al. 2004;Pandey et al. 2020;Payghamzadeh and Kazemitabar 2011;Amente and Chimdessa 2021). However, phenolic compounds are generally present in healthy plant tissues and can accumulate in specialised cell types (Lorenzo et al. 2001;Beckman 2000), and are produced at higher levels and/or released as a defence response, particularly following tissue injury or stress (Beckman 2000;Jones and Saxena 2013). ...
The frequently occurring browning of plant tissue cultures is a considerable problem in causing economic losses. However, the mechanism that causes browning of plant tissues is still controvesial and technologies to effectively prevent browning of plant tissues are still scarce. In the present work, two callus lines derived from grapevine shoot tips (ST-callus) and fruit flesh (F-callus) were used to investigate the relationship between callus-associated endophytes and callus browning. We observed the transfer of browning effects from grapevine brown calli to normal calli, in a contact cocultivation experiment, then detected the emergence of endophytic bacteria from brown calli when the calli were incubated in a microbial culture medium, and the isolates were identified as genus Bacilus. The inoculation of pure cultured B. sp. strain ST-B1 into normal calli at different concentrations caused gradient callus browning, validating the callus browning-causing (CBC) endophyte. In addition, the moderate inhibition of endophytic bacteria in grapevine calli by culturing the calli in ampicillin-containing media reduced the incidence and severity of callus oxidative browning. The experiments were performed separately on two types of grapevine calli, ST-callus (derived from the tip of grapevine shoots), and F-callus (derived from the pulp of grape berries), and gave similar results. The DNA sequence amplicon approach showed that the CBC endophytic bacteria were found in both the normal and brown calli, which differed greatly in their relative abundances. And compared to the normal calli, brown calli greatly reduced the diversity of bacterial endophytes, while the diversity of fungal endophytes between normal and browning calli showed no obvious difference. The work demonstrated that callus-associated endophytes are involved in causing oxidative browning of plant cells, and suggested technologies to minimize the occurrence of the oxidative browning during plant tissue culture practices.
... Whereas walnut trees are recognized as difficult to root (Pei and Gu 2002), the results of cutting have not been satisfactory and there has been no breakthrough in this area to apply it to commercial production (Vahdati et al. 2022). With the maturity and development of tissue culture technology, walnut micropropagation technology has been gained more attention and achieved tremendous progress in rootstock and cultivar propagation (Davis and Sankhla 1988; Gotea et al. 2012;Leslie and McGranahan 1992;Liu et al. 2018;Mc-Granahan et al. 1987;McKenna and Sutter 1996;Payghamzadeh and Kazemitabar 2011;Pei et al. 2007;Yegizbayeva et al. 2021). Rootstock micropropagation for paradox cutting has been successful and used in commercial rootstock production (Ribeiro et al. 2022). ...
Walnut, a woody plant, is regarded as having difficulty rooting when propagated by vegetative methods, such as cutting and layering. A layering experiment was conducted in 2018 and 2022. In 2018, some Juglans species, including J. regia L. seedling (JR), J. regia cv. Liaoning 1 (JR LN1), J. hopeinesis Hu seedling (JH), J. mandshurica Maxim seedling (JM), and J. nigra L. seedling (JN), were the mother plants. The specific research hypotheses were that own-rooted walnut propagule could be obtained through layering. the rooting capacity of different Juglans species would be different, and the rooting ability of JN would be the highest among the samplings. The results indicated that all of these species in the experiment could be rooted by etiolation and indole-3-butyric acid (IBA) treatment and that root occurrence was found 6 to 7 weeks after IBA treatment. The layers (shoots from the mother plant) on the seedlings of JR, JH, and JM obtained rooting percentages (RP) of 75.55%, 84.45%, and 86.67%, respectively, and root numbers (RNs) of 21.8, 42.8, and 38.8, respectively, after 20 days of etiolation and 1% IBA treatment. JR LN1 had difficulty rooting in equal conditions and had a RP of 31.11%. In 2022, JR LN1 was the only mother plant and the IBA concentration was increased to obtain satisfactory RP and RN. With the 4% and 8% IBA treatments, RPs of 88.9% and 93.3% and RNs of 40.3 and 27.7, respectively, were achieved. During the experiment, the RP, RN, root length (RL), and root diameter (RD), as well as the layer height (LH) and layer diameter (LD), were investigated and evaluated. Layers with low vigor were more likely to root, as shown by a nonparametric test conducted for the height and diameter of the layers of the rooting and nonrooting groups. A significantly negative correlation (r = −0.548) was observed between RN and LH. Moreover, the quality of the best results of JR LN1 layering propagule and that with ‘liaoning 1’ 1-year-old seedling were compared. Our results provide more support for the possibility of vegetative propagation of walnut by layering and more information regarding the clonal cultivation of walnut trees and the own-rooted seedling establishment of walnut cultivars.
... Usually, walnut trees are propagated by seed, but high yields are significantly slowed due to their dormant embryos [10]. In vitro regeneration methods using zygotic embryos enable overcoming the barriers in hybridization [11,12], obtaining higher and faster multiplication rates of plants of an elite genotype [10]. ...
... Usually, walnut trees are propagated by seed, but high yields are significantly slowed due to their dormant embryos [10]. In vitro regeneration methods using zygotic embryos enable overcoming the barriers in hybridization [11,12], obtaining higher and faster multiplication rates of plants of an elite genotype [10]. According to Lambardi et al. [13] and Rios Leal et al. [14], in vitro propagation is a method that creates opportunities for obtaining homogeneous plant material with high genetic stability if explants, such as apical buds or shoot tips, are used as initial plant material. ...
Juglans regia L. is a crucial species as a forest tree and for its nutritional and medicinal values. It is also included in the list of endangered species in Albania and thus, there is a need to find methodologies to ensure its rapid regeneration and ex situ conservation. This research, investigated the regeneration of plantlets from synthetic seeds containing shoot tips of four native walnut varieties: ‘Përmet’, ‘Korçë’, ‘Peshkopi’, and ‘Tropojë’. First, in vitro-derived shoot tips from walnut seedlings are encapsulated using sodium alginate. After that, the regeneration potential of the encapsulated shoot tips and the influence of incubation conditions are evaluated. The synthetic seeds were incubated at either 25 °C or 8 °C, with and without dehydration treatment, in 0.5 M sucrose solution for 3 h. The synthetic seeds in both temperature regimes (25 °C and 8 °C) develop plantlets and provid conservation potential without the need for subcultures for 4 and 3.5 months, respectively. Furthermore, all walnut varieties incubated in these conditions achiev a high regeneration rates.
... As was reviewed in [28], contamination is considered a crucial obstacle that prohibits the successful establishment of an aseptic in vitro culture. In this study, contamination rates ranging from 28% to 39% were obtained using sodium hypochlorite. ...
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.
... The establishment of an aseptic in vitro culture is one of the most important factors of plant tissue culture and the effective elimination of contamination contributes to the better development of the explants [26,27]. When introducing P. ponderosa material in vitro, contamination rates ranging from 1% to 16% were obtained using hydrogen peroxide (protocol B). ...
Pinus. ponderosa (P. Lawson and C. Lawson) is a commercial tree and one of the most important forest species in North America. Ponderosa pine suffers hardship when going through vegetative propagation and, in some cases, 15-30 years are needed to achieve full reproductive capacity. Based on previous works on P. ponderosa regeneration through in vitro organogenesis and trying to improve the published protocols, our objective was to analyze the influence of different types of explants, basal culture media, cytokinins, auxins, and light treatments on the success of shoot multiplication and rooting phases. Whole zygotic embryos and 44 µM 6-benzyladenine showed the best results in terms of explants survival. For shoot organogenesis, whole zygotic embryos and half LP (LP medium, Quoirin and Lepoivre, 1977, modified by Aitken-Christie et al., 1988) macronutrients were selected. A significant positive interaction between whole zygotic embryos and half LP macronutrients was found for the percentage of explants forming shoots. Regarding the light treatments applied, a significantly higher percentage of shoots elongated enough to be rooted was detected in shoots growing under blue LED at a light intensity of 61.09 µmol m −2 s −1. However, the acclimatization percentage was higher in shoots previously cultivated under fluorescent light at a light intensity of 61.71 µmol m −2 s −1. Anatomical studies using light microscopy and scanning electron microscopy showed the light treatments promoted differences in anatomical aspects in in vitro shoots; needles of plantlets exposed to red and blue LEDs revealed less stomata compared with needles from plantlets exposed to fluorescent light.
... Compared to other cultivated fruit species, walnut has a more restricted plasticity showing higher requirements with regard to heat, light, humidity and soil types, which are limiting factors in its expansion. Being a very vigorous species, it has high soil nutrient and water requirements, which is why in areas with annual average rainfall below 650-700 mm, irrigation is a mandatory agrotechnical measure [54][55][56][57][58][59][60]. ...
The interest of the countries with traditions of walnut cultivation to increase the quantity and quality of walnut production is due to the fact that the organic cultivation of walnut is a sustainable business, with an ecological, economic and social impact. The walnut plant (Juglans regia) is a resource for food, dermatocosmetic and phytotherapeutic products, is raw material in the wood industry and a source of biomass. It is a species suitable for organic farming, with no chemical factors input (in line with the EU program to reduce pesticide use and fuel consumption by 35% in the context of the current energy crisis), and has properties of atmosphere purification and air-conditioning. Due to the creation of varieties with high production potential, suitable for cultivation in intensive and super-intensive plantations and also due to the government financial support, walnut plantations can contribute to the development of new valorization directions for the obtained production, by processing fruits and other vegetative organs (green nuts, green and dried shells, foliage, timber) with significant profits. In order to establish and capitalize on a walnut plantation in the pedological and climatic conditions of Romania, pedological studies, worthiness and pedoclimatic studies were carried out in the depression area of Gorj County and show the stages of cultivation technology in intensive and super-intensive systems. The sustainability and economic efficiency of setting up organic walnut cultivation has been highlighted in view of the costs for agrotechnical works, labor and material expenses. According to the analysis of the efficiency indicators, it has been shown that the establishment of an ecological walnut orchard, on an area of 1 ha, in Gorj County, Romania, is a sustainable, appropriate and opportune investment, in terms of capitalizing on the agricultural potential of the land, environmental protection and with social and economic impact.
... Temperature can affect AR by influencing water uptake and nutrient metabolism and promoting or inhibiting enzymatic action [127]. There is little research on the effect of temperature on in vitro walnut rooting [128]; however, some coincidences have been reported. Rooting of J. nigra × J. regia 'A35' was greater when induced at 22 • C (60%) than at 27 • C (45%) [102]. ...
Walnuts (Juglans sp.) are allogamous species. Seed-derived plants are not always superior to the selected parent. Clonal propagation of selected stock plants is an essential requirement for the clonal fidelity of the descendants and to maintain their genetic structure. Selection of the desired plant is realized only after reaching maturity, and characterizing and evaluating the performance of adult trees require a long time. Clonal propagation methods ensure proper transmission of characters to descendants and can be used effectively in breeding programs. The commercialization of a cultivar or rootstock depends on the success of vegetative propagation. Walnuts, like other tree species, are
recalcitrant to conventional vegetative propagation methods and even non-conventional in vitro culture (micropropagation). Elucidation of factors determining the success of cloning of desired plants would contribute to understanding current limitations for most genotypes of Juglans. We outline the role of grafting and cuttings and stool layering, as well as in vitro culture on walnut multiplication. These techniques are, in practice, entirely different; nevertheless, they are affected by common factors. The incompatibility of stock-scion and the reduced ability of stem cuttings to root are the main bottlenecks for grafting and cutting, respectively. Genotype, age, and physiological status, reinvigoration or rejuvenation-treatment of donor plant, period of harvesting and processing of explants critically affect the results of methods followed. The in vitro culture technology is the
most suitable for walnut cloning. This also has constraints that affect commercial propagation of most desired genotypes. We describe comprehensive results and synthesis in this review on the asexual reproduction of walnuts, providing a better comprehension of the limiting factors and the ways to overcome them, with direct implications on commercial propagation and the releasing of outstanding genotypes.
... According to these authors, the high level of rooting obtained is not influenced by the type of medium, but by the addition of activated charcoal. Studies carried out by Feyissa, Welander, andNegash (2005), Yakoub-Bougdal, Chérifi, andBonaly (2007) and Kamal and Sayyed (2011) confirmed that the addition of charcoal has positive effects on rooting, not only in species of the genus Dioscorea, but also in other plant species. Activated charcoal is involved in a series of activities, both stimulant and inhibitory, including the release of substances present in its constitution, which, in addition to darkening, promote changes in the composition of the nutrient medium with the adsorption of vitamins, metal ions and plant regulators, including abscisic acid and ethylene, hence favoring the in vitro growth (Sharma, Trivedi, & Purohit, 2012). ...
In vitro oxidation is a problem for some herbaceous and woody species and can cause darkening of tissues and consequently death of explants and plants Therefore, this study aimed to assess the effect of activated charcoal on in vitro yam cultivation, aiming at reducing or eliminating explant oxidation and optimizing the growth of the genotypes Dioscorea alata var. purpurea (Roxb.) A. Pouchet and Dioscorea rotundata Poir. Nodal segments of approximately 1 cm, extracted from plants previously grown in vitro, were introduced into test tubes containing 10 mL of 2GGC culture medium, plus 30 g L-1 sucrose, solidified with 2.2 g L-1 Phytagel® and pH adjusted to 5.8 before autoclaving, containing activated charcoal doses of 0, 1, 2, 3 and 4 g L-1. Plants were maintained for 90 days in a growth room, with temperature of 27 ± 1ºC, photon flux density of 30 μmol m-2 s-1 and photoperiod of 16 hours, after which their development variables were evaluated. Activated charcoal, at the concentration of 4 g L-1 considerably promoted the best development of plants, and the species D. alata var. purpurea showed higher means for all variables studied.
... Moreover, genotype dependence, as well as the low reproducibility of these protocols, have made it's in vitro propagation more complicated. Most of these experiments focused on the browning phenomenon, elongation of shoots or buds, induction of roots, germination of embryos or somatic embryogenesis [6,20,21]. ...
The present investigation was undertaken to standardize the media and the anti-browning regime in order to minimize the phenolic browning of an in vitro culture of Persian walnut cv. Sulaiman. The experiments involved two types of explants, forced and unforced shoot tips, two types of media, Driver and Kuniyuki Walnut (DKW) medium andMurashige and Skooģs (MS) medium, and three types of anti-browning agents, namely, Polyvinylpyrrolidone, ascorbic acid and activated charcoal at 150, 350 and 550 mg/L each. The investigation was replicated thrice under a completely randomized design. Forced shoot tips of cv. Sulaiman on DKW medium showed the best performance in terms of least browning (13.6 ± 10.5%) and highest survival percentage of explants (74.5 ± 2.4%) when treated with ascorbic acid at 550 mg/L. However, unforced shoot tips in MS medium did not perform well and manifested maximum browning (52.9 ± 5.2%). Based on the results, we conclude that incorporation of ascorbic acid in the DKW medium significantly reduced the media and explant browning, thus, it could set the basis of successful in vitro-propagation of walnuts.
... Here, an 84% of plant acclimatization success for the best factor combination was attained. The data here presented and reported by Wang et al. [24] are very good, because, as stated by Payghamzadeh and Kazemitabar [37], it is known that acclimatization is one of the most sensitive steps of in vitro walnut plant production. ...
In vitro micropropagation is already a current multiplication tool for walnut self-rooted cultivars and rootstocks, but walnut grafting is still performed in the field or in greenhouses, mainly using seedlings as rootstocks. The present work describes a new approach to obtain clonal walnut-grafted plants, involving in vitro shoot production of ‘Paradox’ (Juglans hindsi × Juglans regia) cl. ’Vlach’, to be used as rootstock, and J. regia cv. ‘Chandler’, to be used as scion. After completing the in vitro multiplication phase and a seven-day root induction treatment, ‘Vlach’ explants are transferred to ex vitro conditions for root expression while being simultaneously grafted using the in vitro produced ‘Chandler’ scions. The importance of the presence of leaves on both the scion and the rootstock for the success rate of the technique was evaluated. Under optimal conditions, average success rates of 82% for rootstock rooting, 72% for micrografting survival, and 84% for grafted plant acclimatization were achieved. This rooting/grafting combination technique seems able to compete with the traditional techniques of nursery grafting, allowing obtaining high-quality walnut-grafted plants independently of the external weather conditions in a significantly shorter time.
