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High frequency plant regeneration following abnormal shoot organogenesis in the medicinal tree Hovenia dulcis
Abstract
An efficient plant regeneration protocol for shoot organogenesis from Hovenia dulcis callus cultures was established. Induction of organogenic callus was achieved on Murashige and Skoog (MS) medium supplemented
with 4.65μM kinetin and 4.5μM 2,4-dichlorophenoxyacetic acid (2,4-D). Further differentiation of organogenic callus into
primordia, shoot-like structures, and plantlets was achieved on MS medium supplemented with 0.23μM gibberellic acid (GA3) and 0.46μM kinetin. Numerous abnormal shoots developed upon transfer of callus to MS medium containing cytokinins, and
these failed to grow further into whole plantlets. However, transfer of ‘abnormal’ shoots to a fresh MS medium lacking cytokinins
resulted in growth of normal shoots. Elongated shoots subsequently were rooted in basal MS medium, and whole plantlets were
established in a soil mix. Analysis of regenerated plants using random amplified polymorphic DNA (RAPD) confirmed the genetic
stability of these regenerant plantlets.
... Morphological and cytological variation in tissue culture is generally influenced by several factors such as plant genotype, number of subcultures, chemical and physical characteristics of the culture medium, type, age, and treatment of explant, and plant growth regulator application (Bairu et al. 2008;Clarindo et al. 2008;Chen et al. 2009;Jeong et al. 2009;Pérez et al. 2009). The cytological variation expressed via alteration in chromosome number generally results from endopolyploidization (endoreduplication or endomitosis) or nuclear fusion (Borchert et al. 2007;Chen and Gao 2007;Dhooghe et al. 2010;Tang et al. 2010). ...
... These variations are heritable, i.e., transmitted through meiosis, and are usually irreversible. The main factors that influence the variation generated from tissue culture are: (1) the degree of departure from organized growth, (2) the genotype, (3) the culture medium, (4) growth regulators, and (5) the tissue source (Karp 1995;Borchert et al. 2007;Bairu et al. 2008;Clarindo et al. 2008;Chen et al. 2009;Jeong et al. 2009;Pérez et al. 2009;Tang et al. 2010). Such variation can be assessed by analysis of the phenotype, chromosome number and structure, proteins, or direct DNA evaluation of plants ( De Klerk 1990;Borchert et al. 2007;Clarindo et al. 2008;Jeong et al. 2009;Pérez et al. 2009), via morphological observation, root tip chromosome counting, flow cytometry, random amplified polymorphic DNA (RAPD), AFLP banding patterns, or other techniques (Clarindo et al. 2008;Jeong et al. 2009;Pérez et al. 2009;Mallón et al. 2010;Tang et al. 2010). ...
... The main factors that influence the variation generated from tissue culture are: (1) the degree of departure from organized growth, (2) the genotype, (3) the culture medium, (4) growth regulators, and (5) the tissue source (Karp 1995;Borchert et al. 2007;Bairu et al. 2008;Clarindo et al. 2008;Chen et al. 2009;Jeong et al. 2009;Pérez et al. 2009;Tang et al. 2010). Such variation can be assessed by analysis of the phenotype, chromosome number and structure, proteins, or direct DNA evaluation of plants ( De Klerk 1990;Borchert et al. 2007;Clarindo et al. 2008;Jeong et al. 2009;Pérez et al. 2009), via morphological observation, root tip chromosome counting, flow cytometry, random amplified polymorphic DNA (RAPD), AFLP banding patterns, or other techniques (Clarindo et al. 2008;Jeong et al. 2009;Pérez et al. 2009;Mallón et al. 2010;Tang et al. 2010). ...
... Morphological and cytological variation in tissue culture is generally influenced by several factors such as plant genotype, number of subcultures, chemical and physical characteristics of the culture medium, type, age, and treatment of explant, and plant growth regulator application (Bairu et al. 2008;Clarindo et al. 2008;Chen et al. 2009;Jeong et al. 2009;Pérez et al. 2009). The cytological variation expressed via alteration in chromosome number generally results from endopolyploidization (endoreduplication or endomitosis) or nuclear fusion (Borchert et al. 2007;Chen and Gao 2007;Dhooghe et al. 2010;Tang et al. 2010). ...
... These variations are heritable, i.e., transmitted through meiosis, and are usually irreversible. The main factors that influence the variation generated from tissue culture are: (1) the degree of departure from organized growth, (2) the genotype, (3) the culture medium, (4) growth regulators, and (5) the tissue source (Karp 1995;Borchert et al. 2007;Bairu et al. 2008;Clarindo et al. 2008;Chen et al. 2009;Jeong et al. 2009;Pérez et al. 2009;Tang et al. 2010). Such variation can be assessed by analysis of the phenotype, chromosome number and structure, proteins, or direct DNA evaluation of plants ( De Klerk 1990;Borchert et al. 2007;Clarindo et al. 2008;Jeong et al. 2009;Pérez et al. 2009), via morphological observation, root tip chromosome counting, flow cytometry, random amplified polymorphic DNA (RAPD), AFLP banding patterns, or other techniques (Clarindo et al. 2008;Jeong et al. 2009;Pérez et al. 2009;Mallón et al. 2010;Tang et al. 2010). ...
... The main factors that influence the variation generated from tissue culture are: (1) the degree of departure from organized growth, (2) the genotype, (3) the culture medium, (4) growth regulators, and (5) the tissue source (Karp 1995;Borchert et al. 2007;Bairu et al. 2008;Clarindo et al. 2008;Chen et al. 2009;Jeong et al. 2009;Pérez et al. 2009;Tang et al. 2010). Such variation can be assessed by analysis of the phenotype, chromosome number and structure, proteins, or direct DNA evaluation of plants ( De Klerk 1990;Borchert et al. 2007;Clarindo et al. 2008;Jeong et al. 2009;Pérez et al. 2009), via morphological observation, root tip chromosome counting, flow cytometry, random amplified polymorphic DNA (RAPD), AFLP banding patterns, or other techniques (Clarindo et al. 2008;Jeong et al. 2009;Pérez et al. 2009;Mallón et al. 2010;Tang et al. 2010). ...
Anthurium anther culture was successfully established using half-anthers as explants. Explants were cultured on Winarto–Teixeira basal
medium (WT-1) containing 0.01mg/l α-naphthalene acetic acid (NAA), 0.5mg/l thidiazuron (TDZ), and 1.0mg/l 6-benzylaminopurine
(BAP), or on New Winarto–Teixeira basal medium (NWT-3) supplemented with 0.02mg/l NAA, 1.5mg/l TDZ, and 0.75mg/l BAP for
callus initiation. Regenerated calli produced multiple shoots on WT-1, which were then rooted in NWT-3 supplemented with 1%
activated charcoal. Plantlets were acclimatized ex vitro using a mixture of burned rice husk, rice husk, and bamboo peat (1:1:1,
v/v/v) as the potting medium. There was considerable morphological and cytological diversity of regenerants derived from anther
culture, which are described in detail in this study. The callus cluster color ranged from green to light green and had a
high regeneration capacity (7.3 and 4.8 shoots/callus cluster), light reddish-yellow callus showed moderate regeneration (2.6
shoots/callus cluster), while reddish-yellow callus had the lowest regeneration capacity (1.5 shoots/callus cluster). Morphological
variations clearly observed in regenerants derived from this technique included alterations in plant size, peduncle length,
spathe position compared to leaves, the type and number of buds, spathe and spadix color, and spadix length. There were also
cytological variations in both in vitro and ex vitro regenerants of anther culture with 23–29% haploids, 5–10% aneuploids,
56–69% diploids, and 3–4% triploids. The results strengthen other studies in which the development of anther cultures, especially
via callus formation, resulted in morphological and cytological alterations. These variations have been discussed to great
length in this paper.
KeywordsAnther culture–Morphology–Cytology–Somaclonal variation–
Anthurium
... So far, the methods of plant tissue culture developed for H. dulcis aim at the establishment of protocols for plant propagation, including phytochemical analysis of in vitro plants (Yang et al. 2013;Echeverrigaray et al. 1998;Eom et al. 2002;Jeong et al. 2009;Park et al. 2012;Li et al. 2006;Ribeiro et al. 2010). Friable calli were previously reported for the species, but only in association with plant regeneration (Park et al. 2012;Jeong et al. 2009). ...
... So far, the methods of plant tissue culture developed for H. dulcis aim at the establishment of protocols for plant propagation, including phytochemical analysis of in vitro plants (Yang et al. 2013;Echeverrigaray et al. 1998;Eom et al. 2002;Jeong et al. 2009;Park et al. 2012;Li et al. 2006;Ribeiro et al. 2010). Friable calli were previously reported for the species, but only in association with plant regeneration (Park et al. 2012;Jeong et al. 2009). Also, non-organogenic compact callus cultures were established (Ribeiro et al. 2010(Ribeiro et al. , 2015. ...
Medicinal plants are an important therapeutic option for a large share of the world’s population. To establish an in vitro culture system for the production of secondary metabolites from Hovenia dulcis, we studied the effect of auxins, cytokinins, absence of light, and silver nitrate on the development of friable callus. Callus cultures were established for the first time and used to obtain cell suspension cultures. Supplementation with KIN (Kinetin) produced calli with both compact and friable areas, while the addition of TDZ (Thidiazuron) only produced compact callus. The maintenance of cultures in the dark induced a slight enhancement on friability when the auxin PIC (Picloram) was present in the culture medium. The addition of silver nitrate promoted the formation of friable calli. Dry weight analysis showed no significant differences in biomass growth, and, therefore, 2.0 mg.L-1 was considered the most suitable treatment. The presence of silver nitrate was not required for the establishment of cell suspension cultures. Dry weight analysis of cell suspensions showed higher biomass production in the absence of silver nitrate. PIC promoted 100% of cell suspension culture formation in the absence of silver nitrate, and higher biomass production was observed with the lowest concentration (0.625 mg.L-1). No morphological differences were observed among the different concentrations of PIC. Phytochemical screening showed the presence of saponins, flavonoids, flavonols and catechins in the extracts obtained from H. dulcis calli. These results show that the cell cultures herein established are potential sources for the production of H. dulcis secondary metabolites of medicinal interest.
... The in vitro propagation of H. dulcis was previously evaluated through protocols of indirect organogenesis (Jeong et al. 2009), indirect somatic embryogenesis (Yang et al. 2013) and direct organogenesis (Echeverrigaray et al. 1998;Park et al. 2006). In these studies, use of the cytokinins BA and KIN also proved to be efficient for inducing morphogenic response. ...
... Although cytokinins are known to regulate morphogenesis in plant tissue culture, stimulating shoot proliferation, their presence can inhibit the elongation process (Schaller et al. 2015). Our results corroborate previous studies with H. dulcis (Jeong et al. 2009) when shoots obtained by indirect organogenesis were elongated and rooting was performed on growth regulator-free medium. However, supplementation with auxins was necessary to induce these morphogenic responses in three other studies on the species (Echeverrigaray et al. 1998;Park et al. 2006;Yang et al. 2013), as well as species of the genus Zizyphus (Goyal and Arya 1985;Rathore et al. 1992;Mathur et al. 1995) from the Rhamnaceae family. ...
This study describes in vitro propagation and cryopreservation of Hovenia dulcis, a woody species used in traditional medicine. Stem and leaf explants from axenic seedlings were cultivated on Murashige and Skoog (MS) medium containing 6-benzyladenine (BA) and kinetin (KIN) alone or in combination (0.1, 0.2, 0.5 mg L⁻¹). For in vitro propagation, rates of regeneration (percentage of responsive explants) and proliferation (multiplication capacity of explant-derived shoots) were evaluated after 30 days and five subcultures, respectively. For cryopreservation by V Cryo-plate technique, shoot tips were excised from microcuttings cultured from in vitro-grown stock plants, or excised directly from axillary shoots of stock plants. The shoot tips were precultured in 0.3 M sucrose (24 h), exposed to loading (20 min) and to PVS2 (0–150 min) before storage in liquid nitrogen. The regrowth was assessed by plating of shoot tips on recovery medium (MS with BA + KIN), with or without a sterile filter paper over the culture medium. Cryopreservation was evaluated by survival (4-weeks) and recovery (8-weeks). The highest regeneration by direct organogenesis (100%) were reached on medium with BA + KIN (0.5 mg L⁻¹ each). Shoots maintained multiplication capacity, showing the highest proliferation (87%) in the presence of BA. Shoot elongation and rooting were achieved on growth regulator-free MS. The most efficient cryopreservation protocol (68% survival and 62% recovery) applied exposure to PVS2 (120 min), and recovery on medium containing BA + KIN (0.5 mg L⁻¹ each) with filter paper. The propagation and cryopreservation of H. dulcis may contribute to its conservation and that of other woody species.
... In the present study, friable calli were observed when 2,4-D and PIC were added to the culture medium, while only NAA induced compact callus which is in contrast with Jeong et al. (2009) findings, where 2,4-D promoted the formation of compact callus in leaf explants. Similar results were reported for other species such as Gossypium hirsutum, where 2,4-D promoted only friable callus while NAA was responsible for compact callus formation (Baksha et al., 2006). ...
... Compact callus cultures obtained on the present study were subcultivated for at least 6 months and no morphological changes were observed. These results are highly desirable, since Jeong et al. (2009) observed oxidation in H. dulcis non-organogenic calli after 20 days. ...
Plant biotechnology enables the production of biomass under controlled conditions, providing the synthesis of raw material in a continuous and homogeneous way. The aim of the present study was to establish Hovenia dulcis callus cultures and to evaluate their antioxidant potential in comparison with wild grown and in vitro plants. The best results for compact calli were obtained with the supplementation of 1-naphthaleneacetic acid (NAA) at 2.5 mg L-1 and the use of benzylaminopurine (BAP) enhanced callus growth. The medium supplemented with 2.5 mg L-1 NAA + 0.65 mg L-1 BAP produced 115.3±28.2 mg of dry weight. The auxin NAA was responsible for the production of light-green compact callus, while picloram and 2,4-D promoted mixed (friable and compact) calli. Total polyphenols and total flavonoids were found in higher concentrations in wild grown plants, whereas the reduction capacity and DPPH radical scavenging assays recorded higher antioxidant activity in calluses extracts. The protocols established here represent a viable and effective way for producing substances with medicinal interest.
... The morphogenetic pattern associated with the induction and proliferation of NCs observed in the present study culminated with the large-scale development and regeneration of elongated microshoots. These features appear to be recurrent in the in vitro culture of V. reitzii and were also described to this genus in other studies (Rech Filho et al., 2005, 2009Alves et al., 2006;Guerra and Dal Vesco, 2010). According to these authors, the coloration pattern and texture was modulated by the composition of the culture medium employed in the induction phase. ...
... Differences in in vitro regenerative potential of adventitious shoots may be attributed to the species characteristics, the regenerative system, and culture medium composition (George and Debergh, 2008;Preece, 2008). In Hovenia dulcis, high regenerative rates of adventitious buds from nodular callus were obtained on MS culture medium supplemented with 2-iP, and the subsequent development of microshoots was obtained with their transfers to medium free of PGR (Jeong et al., 2009). In S. birrea high regenerative rate of adventitious shoots was observed from meristematic nodules in response to the WPM culture medium supplemented with BAP combined with NAA, IBA or IAA (Moyo et al., 2009). ...
Micropropagation systems based on nodular cultures (NCs), are considered as an intermediary in vitro morphogenetic route, diverging from regenerative systems based on organogenesis and somatic embryogenesis. The aim of this study was to establish a regenerative protocol based on the induction and development of NCs in Vriesea reitzii, an endangered bromeliad from the Atlantic forest which also has ornamental value. Additionally structural analyses were performed in order to better understand this in vitro morphogenetic route. NCs were regenerated in MSB culture medium free of PGR or supplemented with different levels of NAA alone or in combination with in combination with 2-iP. The subculture of these NCs on MSB medium supplemented with 10 μM of GA3 promoted the synchronized shoot elongation. A regenerative efficiency of 12.4 g g−1 of NCs was obtained, and this results in 5300 microshoots after 10 weeks in culture. The structural analyses of the NCs revealed that the regenerative process occurs from the proliferation of meristematic cell groups resulting in the development of multiple shoot meristems and buds. The development of NCs leads to the formation of monopolar structures called microshoots, which evolve to elongated shoots. Intermediary features shown in NCs are consistent with their classification as an intermediary system among organogenesis and somatic embryogenesis.
... Mostly explants derived from seedlings have been used for callus-mediated organogenesis in medicinal tree species (Table 1). For instance, Jeong et al. (2009) reported callus-mediated organogenesis in Hovenia dulcis from leaf explants obtained from seedlings. More recently, Jaiswal et al. (2021) used leaf explants excised from in vitro derived shoots for callus induction, shoot proliferation and plant regeneration in Spondias pinnata. ...
Many tree species are an excellent source of a wide range of bioactive compounds of pharmaceutical importance. However, overexploitation of medicinal trees to fulfil the demand for plant-based herbal drugs puts pressure on its natural population. Therefore, many tree species are declining continuously, particularly those used in pharmaceutical industries. In addition, limited production of secondary metabolites from a specific part and only at a particular developmental stage and sometimes very-low yield are some major bottlenecks when secondary metabolites are isolated directly from a tree species. Tissue culture-based biotechnological interventions for propagation, in vitro conservation and secondary metabolite production in medicinally important tree species have been practiced during the last two–three decades. Many medicinally important trees successfully propagated in vitro through different modes of regeneration i.e., axillary shoot proliferation, adventitious organogenesis and somatic embryogenesis. Success in in vitro propagation of most of the medicinal trees has been achieved through axillary shoot proliferation. Recent studies on medicinal trees showed that ex vitro rooting is an ideal method of rooting of microshoots. Gene targeted molecular markers have now been preferred for genetic fidelity of tissue culture-raised plants of medicinal trees. In recent years, newly developed droplet-vitrification and cryo-plate methods increased the applicability of cryopreservation for the long-term conservation of many medicinal trees. Several bioactive compounds of pharmaceutical importance are produced from trees via in vitro culture technique. There are a few success stories of producing secondary metabolites at a commercial scale from medicinal trees i.e., taxol, camptothecin and azadirachtin. This review paper presents the recent progress on plant tissue culture-mediated biotechnological advances in medicinal trees, emphasizing different aspects of in vitro propagation, conservation, and production of bioactive compounds of pharmaceutical importance.
... For genetic integrity, DNA-based molecular markers have been proposed as an excellent tool for identifying geographical variation, genetic diversity, phylogenetic relationship and authentication of plant species, pharmacognostic characterization, species characterization and genetic mapping in medicinal plants (Joshi et al. 2004). Molecular marker techniques such as inter simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) analysis have been used to assess variability or similarity within or between the plants derived from plant tissue culture and the donor mother plant (Rout et al. 2009;Jeong et al. 2009;Jain et al. 2011). ...
Corallocarpus epigaeus (Rottler) Hook.f. is an endangered tuberous medicinal climber of family Cucurbitaceae. Despite high medicinal value, over-exploitation made it threatened. In vitro propagation has been adopted for conserving this endangered medicinal plant. Direct shoots induction was achieved from nodal explants on MS medium fortified with various concentrations of BAP and TDZ individually and BAP + IAA, TDZ + IAA, BAP + l-glutamic acid and TDZ + l-glutamic acid combinations. The highest frequency of multiple shoots (43.33 ± 0.53) was achieved on MS medium fortified with 1.5 mg/l TDZ + 1.5 mg/l IAA from nodal explants but shoot length (12.9 ± 0.15 cm) was high on MS medium supplemented with 1.0 mg/l TDZ and 2.0 mg/l l-Glutamic acid. The highest percentage (78%) of rooting was achieved on half strength MS medium augmented with 1.0 mg/l IBA with a mean number of roots 10.76 ± 0.30 cm, an average root length is 1.69 ± 0.07 cm. Rooted plantlets were acclimatized in the greenhouse and successfully transplanted to natural conditions with a 68% survival rate. ISSR markers were used to check the genetic fidelity between in vivo and in vitro developed plantlets. The results indicated that the micropropagated plants are monomorphic and true type when compare with mother plant.
... The plants have developed several strategies in order to cope with various extracellular stresses, these strategies usually based on some steps such as recognition of the stress condition, signal transduction and regulation of gene expression leading to adaptive or protective physiological responses (2). Cytological changes in tissue culture are affected by many factors such as ionizing radiation, type of media, extent of subculture, choice of plant hormones and responses to abiotic factors such as salinity (3). Somatic Embryogenesis Receptor-Like Kinase (SERK) and genes encode leucine rich repeat receptor-like kinases (LRR-RLKs) are expressed in all organs of the plant (4). ...
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... Some investigators have noted that indirect regeneration of plantlets requires a longer induction. The growing medium is usually supplemented with cytokinins (Howell et al., 2003) and usually results in variability among the regenerating plantlets (Mondal et al., 2004;Pontaroli and Camadro, 2005;Bairu et al., 2006;Park et al., 2006;Jeong et al., 2009). Genetic changes may occur at cellular and (more frequently) at ploidy levels such as in chromosome structure (Radic et al., 2005) or at molecular levels with punctual mutations in DNA (Chen et al., 2012) The cause of somaclonal variation in higher plants has been reported during different biochemical and molecular events including changes in DNA methylation pattern, activation of transposable elements and chromosome remodeling (Hirochika, 1993;Price et al., 2002). ...
The objective of this study was to induce a rapid as well as prolific shoot regeneration protocol for micropropagation and RAPD analysis of Guizotia abyssinica Cass. which is an important herbaceous plant of immense industrial value via direct and indirect organogenesis from apical bud, axillary bud, leaf and internode explants. Best seed germination was obtained on cotton irrigated with liquid MS medium. Out of the four explants used, apical bud proved to be the best in terms of shoot regeneration and multiplication. Best shoot multiplication was obtained from apical bud, axillary bud and leaf explants on MS medium supplemented with 2.22 μM BAP + 2.85 μM IAA. Whereas supplementation of MS medium with 2.22 μM BAP + 28.55 μM IAA produced maximum number of shoots from internode explants. BAP (0.44 μM) in combination with Kn (0.46 μM) proved suitable for maximum mean shoot length. Moreover, culturing the regenerated shoots on half-strength liquid MS medium supplemented with NAA (2.68 μM) induced maximum rooting from elongated shoots (direct and indirect regeneration). The plantlets were established in plastic cups containing vermiculite, soil, sand and farm yard manure and then successfully transferred to field with 97.33% survival. Analysis of RAPD recognized 197 different amplification products and showed the presence of somaclonal variation in the plantlets arising from direct regeneration as well as from indirect regeneration. The protocol developed in this study is suitable for propagation of quality planting material for commercialization, germplasm conservation and for future genetic improvement studies.
... Few attempts were made on in vitro regeneration of B. diffusa using leaves and stem (nodes and shoot tip) as explants (Sudarshana et al. 2008;Wesely et al. 2010;Saini et al. 2011,) but the protocol for genetic integrity has not been established of the micropropagated plants of B. diffusa L. DNA-based molecular markers have been proposed as an excellent tool for identifying geographical variation, genetic diversity, phylogenetic relationship, authentication of plant species, pharmacognostic characterization, species characterization and genetic mapping in medicinal plants (Joshi et al. 2004). Molecular marker techniques, such as random amplified polymorphic DNA (RAPD) analysis, have been used to assess variability or similarity within or between the plants derived from plant tissue culture and the donor plant (Jeong et al. 2009;Jain et al. 2011). RAPD is a simple, largely automatable technique which requires only a small amount of DNA and can be performed without the use of radioactivity (Williams et al. 1990). ...
Boerhaavia diffusa L is a medicinal herb with immense pharmaceutical significance. The plant is used by many herbalist, Ayurvedic and pharmaceutical industries for production biopharmaceuticals. It is among the 46 medicinal plant species in high trade sourced mainly from wastelands and generally found in temperate regions of the world. However, the commercial bulk of this plant shows genetic variations which are the main constraint to use this plant as medicinal ingredient and to obtain high value products of pharmaceutical interest from this plant. In this study, we have regenerated the plant of Boerhaavia diffusa L through nodal explants and evaluated genetic fidelity of the micropropagated plants of Boerhaavia diffusa L with the help of random amplified polymorphic DNA (RAPD) markers. The results obtained using RAPD showed monomorphic banding pattern revealing genetic stability among the mother plant and in vitro regenerated plants of Boerhaavia diffusa L.
... In the present investigation, it was observed that the total number of regenerants increased as the concentrations of TDZ increased in combination with 0.5 mg/L GA 3 or 0.25 mg/L NAA. This was in line with the observations on differentiation of organogenic callus into plantlets in Hovenia dulcis [33] on MS medium supplemented with 0.23 µM gibberellic acid and 0.46µM kinetin. Similarly maximum number of shoots was achieved from leaf section callus of Hyptis suaveolens [34] on MS medium containing 0.5 mg/L BA and 0.5 mg/L GA 3 . ...
Shoot organogenesis and plant regeneration were achieved on callus derived from leaf section and stem base explants of Quisqualis indica (Combretaceae). In vitro cultures were established using nodal segments obtained from mature field-grown shrubby plants. For the development of optimized protocol, different types and concentrations of plant growth regulators were used to induce adventitious shoot regeneration via callus from leaf section and one-node stem base explants obtained from in vitro regenerated micro shoots and direct field-grown newly flush-off shoots. The TDZ was considered to be the best among the cytokinins (6-benzyladenine (BA), 6-(?-?, dimethylallyamino purine) (2-iP) and thidiazuron (TDZ) added to the Murashige and Skoog’s medium (MS) for adventitious shoot productions. A combination of 1.0 mg/L TDZ and 0.5 mg/L GA3 was most effective in stimulating callus induction and adventitious shoot regeneration from the leaf section derived calli with an average of 6 shoots per callus explant and an average of 8 shoots per callus explant originated from one-node stem base explants. In vitro raised shoots were sub-cultured on MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L GA3 for further shoot growth. Maximum rooting of in vitro regenerated shoots was obtained on MS medium supplemented with either 0.5 mg/L indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) individually or a combination of 0.5 mg/L IAA and 0.5 mg/L IBA. Plantlets raised in vitro were acclimatized and subsequently transferred to experimental field.
... In the present investigation, it was observed that the total number of regenerants increased as the concentrations of TDZ increased in combination with 0.5 mg/L GA 3 or 0.25 mg/L NAA. This was in line with the observations on differentiation of organogenic callus into plantlets in Hovenia dulcis [33] on MS medium supplemented with 0.23 µM gibberellic acid and 0.46µM kinetin. Similarly maximum number of shoots was achieved from leaf section callus of Hyptis suaveolens [34] on MS medium containing 0.5 mg/L BA and 0.5 mg/L GA 3 . ...
Shoot organogenesis and plant regeneration were achieved on callus derived from leaf section and stem base explants of Quisqualis indica (Combretaceae). In vitro cultures were established using nodal segments obtained from mature field-grown shrubby plants. For the development of optimized protocol, different types and concentrations of plant growth regulators were used to induce adventitious shoot regeneration via callus from leaf section and one-node stem base explants obtained from in vitro regenerated micro shoots and direct field-grown newly flush-off shoots. The TDZ was considered to be the best among the cytokinins (6-benzyladenine (BA), 6-(-, dimethylallyamino purine) (2-iP) and thidiazuron (TDZ) added to the Murashige and Skoog's medium (MS) for adventitious shoot productions. A com-bination of 1.0 mg/L TDZ and 0.5 mg/L GA 3 was most effective in stimulating callus induction and adventitious shoot regeneration from the leaf section derived calli with an average of 6 shoots per callus explant and an average of 8 shoots per callus explant originated from one-node stem base explants. In vitro raised shoots were sub-cultured on MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L GA 3 for further shoot growth. Maximum rooting of in vitro regenerated shoots was obtained on MS medium supplemented with either 0.5 mg/L indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) individually or a combination of 0.5 mg/L IAA and 0.5 mg/L IBA. Plantlets raised in vitro were acclima-tized and subsequently transferred to experimental field.
... Development of an advanced propagation technology such as somatic embryogenesis might be an efficient method for biotechnological improvement of H. dulcis. In vitro 2 The Scientific World Journal propagation of H. dulcis by organogenesis has been reported [10]. Our present study describes methods to obtain normal SEs and plantlet conversion following secondary somatic embryogenesis via culture of H. dulcis in agar and liquid media. ...
Embryogenic callus was obtained from mature seed explants on medium supplemented with 2,4-dichlorophenoxyacetic acid. Primary somatic embryos (SEs) can only develop into abnormal plants. Well-developed SEs could be obtained through secondary somatic embryogenesis both in solid and liquid cultures. Temperature strongly affected induction frequency of secondary embryogenesis. Relatively high temperature (30°C) and germinated SEs explants were effective for induction of secondary somatic embryos, and low temperature (20°C) was more suitable for further embryo development, plantlet conversion, and transplant survival. Somatic embryos formed on agar medium had larger cotyledons than those of embryos formed in liquid medium. Supplementing 0.1 mg L(-1) 6-benzyladenine (BA) was effective for plant conversion; the rate of plant conversion was 43.3% in somatic embryos from solid culture and 36.5% in embryos from liquid culture. In vitro plants were successfully acclimatized in the greenhouse. The protocol established in this study will be helpful for large-scale vegetative propagation of this medicinal tree.
... In the present study, the callus-derived plants after 4 years showed no polymorphism, which may be due to the genetic stability of the plant genome. No variability in long-term callus-derived progenies has been reported in Citrus sinensis (Khan et al. 2009), Hovenia dulcis (Jeong et al. 2009), and Centaurea ultreiae (Mallón et al. 2010). However, in contrast, the instability of the plants regenerated through organogenesis has also been documented in Asparagus officinalis (Pontaroli and Camadro 2005) and Codonopsis lanceolata (Guo et al. 2006). ...
Trichopus zeylanicus subsp. travancoricus (known as Arogyapacha), an endangered ethnomedicinal plant of the Western Ghats of South India, serves as the major source
of the commercial drug Jeevani. The present study established a long-term high frequency in vitro propagation protocol for Arogyapacha. Callus obtained from
the branch–petiole explants cultured on Murashige and Skoog (MS) medium with 4.5μM 2,4-dichlorophenoxyacetic acid upon subculture
to medium with different concentrations of 6-benzyladenine (BA) either alone or in combination with an auxin favoured shoot
morphogenesis. Medium with 13.3μM BA alone facilitated high frequency shoot bud (mean of 93.2) formation. Medium with lower
concentrations of BA (4.4, 6.6 and 8.8μM) alone or in combination with lower concentration of α-naphthaleneacetic acid (NAA)
or indole-3-butyric acid (IBA) favoured better shoot growth than 13.3μM BA containing medium, but with reduced number of
shoot buds. Subsequent cultures on medium with lower concentrations of BA and also on MS basal media facilitated shoot formation
as well as growth of shoots. The shoot regeneration potential showed no decline up to 5years. Culture of the in vitro-derived
whole branch–leaf explants on MS basal medium developed shoots directly from the node. On medium with 19.6μM IBA, the whole
branch–leaf explants induced nodular callus from the node, which developed shoots later. Subsequent cultures on medium with
BA exhibited high frequency shoot formation. The transfer of shoots after 10–15days culture on half-strength MS medium containing
2.7μM NAA to half-strength basal medium induced a mean of 11.3 roots. Field survival of plantlets relied on the soil mix:
a 1:4 ratio of sand and red-soil exhibited the highest plantlets survival (86.6%). RAPD profile of the source plant and plants
regenerated from calli after 4years showed no polymorphism. The established plantlets with morpho-floral features similar
to that of the source plants flowered normally and set fruits.
KeywordsArogyapacha–Jeevani–Organogenesis–RAPD
... Several techniques such as cytological, isozymes and molecular markers have been employed to detect variation if any or to confirm the genetic stability of micropropagated plants (Gupta and Varshney, 1999). Usefulness of RAPD in detection of variation in micropropagated plants has been amply demonstrated in large array of plants viz., Loblolly pine (Tang, 2001), Chestnut hybrids (Carvalho et al., 2004), Chlorophytum arundinaceum (Lattoo et al., 2006), Hovenia dulcis (Jeong et al., 2009), Hypericum perforatum (Goel et al., 2009) and Platanus (Sun et al., 2009) In this paper, we report an efficient regeneration system via direct multiple shoot bud induction from leaf explants of P. cablin, the effect of the origin and age of the donor plant, and physiological age of leaf on adventitious shoot proliferation and plant regeneration. An attempt has been made to assess the clonal fidelity of micropropagated plants by RAPD analysis and to verify essential oil profiles by gas-chromatographic (GC) analysis. ...
Rapid and prolific shoot regeneration via direct organogenesis was induced from leaf explants of Patchouli, Pogostemon cablin (Blanco) Benth., an aromatic plant of immense industrial value, on Murashige and Skoog (MS) medium with benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA) within 4 weeks. The adventitious shoot bud induction and plant regeneration were greatly influenced by the origin, age of donor plant, and leaf position on stem. Leaf explants prepared from in vivo plants of different ages showed higher regeneration response as compared to the explants from in vitro plantlets of respective age. The shoot regeneration ability of explants was significantly related to the age of the donor plants as well as the leaf position on the stem. The highest number of shoots (94.6/explants) was obtained from 96.2% of leaf explants derived from the leaves located on the second node of the 3-month-old in vivo plants on Murashige and Skoog's (MS) medium containing 2.5 μM benzylaminopurine (BAP) and 0.5 μM naphthaleneacetic acid (NAA). Incorporation of 1.0 μM gibberellic acid (GA3) in MS medium significantly improved the shoot elongation (1.8-fold) within 2 weeks in 95% of the cultures. Regenerated shoots rooted spontaneously on growth regulator-free half strength MS medium and were successfully hardened and transferred to nursery with 96–100% survival rate. Genetic fidelity of the in vitro derived plants was assessed using random amplified polymorphic DNA (RAPD). Fourteen arbitrary decamers displayed same banding profile within all the micropropagated plants and in vivo explant donor plant. The molecular analysis complemented and compared well and showed genetic stability in the plants regenerated through direct shoot bud differentiation from leaf explants. The gas chromatogram of the extracted oils from in vitro derived plants and the mother stock plant showed similar essential oil profiles. Rapid and high multiplication frequency, molecular, genetic and essential oil content stability ensure the efficacy of the protocol developed for the production of this industrially important aromatic plant.
The low rate of Dendrobium regeneration through seed culture is a significant limitation of mass propagation development in new hybrids. An efficient protocol of in vitro regeneration through pseudobulb segment culture has been established for Dendrobium ‘Danny Dame’. Leaves and roots of seven-month-old seedlings were detached from the pseudobulb. Unsegmented pseudobulb and segments of apical, medial, and basal excised from seedlings. The four types of explants were cultured on Murashige and Skoog (MS) basal medium supplemented with different combinations of 1-naphthaleneacetic acid (NAA) and 6-benzyl amino purine (BAP). The highest number of shoots was observed in 1 mg L-1 BAP and 0.1 mg L-1 NAA using unsegmented pseudobulb explant after 2 months of culture. The unsegmented pseudobulb had a higher survivability rate and the number of shoots per explant than segmented pseudobulbs. However, if accumulated, segmented pseudobulbs can produce the number of shoots 2-4 times more than unsegmented pseudobulbs. The segment that had the highest average number of shoots was the basal segment. Two types of shoots were observed, both single shoot and multi shoot. The new shoots formation was developed from protuberance and or axillary bud. Roots were kept to grow only on the basal part of unsegmented explants and basal segment.
Apple (Malus domestica) is a delicious fruit of high economic value. However, numerous destructive fungi affect apples, greatly limiting their production. Many studies have shown that salicylic acid and jasmonic acid improve plant resistance to fungal diseases, but the potential mechanisms of cytokinin action and fungal diseases remain largely unreported. The IPT gene family encodes the proteins required for cytokinin biosynthesis and plays a central role in plant biological progresses. In our previous study, we found that the disease resistance in autotetraploid apple was much better than ‘Hanfu’ diploid and analyzed the transcriptome data between ‘Hanfu’ diploid and autotetraploid apple. We found that the MdIPT8 gene was significantly differentially expressed. Therefore, we focused on gene family functions in cytokinin biosynthesis and biotic stress responses. We identified ten IPT genes in the ‘Hanfu’ whole genome that are unevenly distributed across the five apple chromosomes. Phylogenetic analysis indicated that this gene family belonged to two groups. Transcriptional analysis of the ten IPT genes indicated that MdIPT8 could be induced under Colletotrichum gloeosporioides infection in apple leaves. The MdIPT8 protein, when fused to the green fluorescent protein, identified the chloroplast as the site of protein localization. Finally, we confirmed that overexpression of MdIPT8 increased the resistance of C. gloeosporioides infection. Taken together, we have identified that the endogenous cytokinin content of ‘Hanfu’ leaves increased during C. gloeosporioides infection and the application of exogenous cytokinin improved the resistance of apple leaves to C. gloeosporioides and found that MdIPT8 can increase apple anthracnose resistance by improving the biosynthesis of cytokinin.
Protocol for in vitro propagation of Chamaecyparis obtusa was established. Adventitious shoots were initiated from epicotyl explants after 4 weeks of culture on MS medium supplemented with 3% (w/v) sucrose and several concentrations of BAP. The effects of various nutritive media, carbon sources and several concentrations (4.44 to 22.20 mu M) of BAP on in vitro shoot (upside of plantlet excepted roots) elongation and multiplication were determined. Optimal shoot growth was achieved on Woody Plant (WP) medium, they have over 25 shoots and 2.5 growth index (GI) after 8 weeks of culture. Among the carbon sources tested, 1% (w/v) fructose showed the highest length of the shoots (appr. 2.2 GI). The highest adventitious shoot induction was obtained on 4.44 mu M of BAP, which gave 6.7 number of shoots after 8 weeks of culture and maximum shoot elongation was observed on WP basal medium without BAP which gave 7.8 cm long shoot 8 weeks after the transfer.
This study was conducted to examine effects of plant growth regulators and light resources on the formation of multiple shoot and plant regeneration of Hovenia dulcis var. koreana Nakai. Stem and shoot tip were cultured on MS medium or WPM supplemented with various plant growth regulators. At the single treatment, the highest shoot formation was obtained when stem explants were cultured on WPM supplemented with kinetin . MS medium containing NAA 0.1 and TDZ gave the best results for shoot induction rate and shoot growth in combination treatments. Of the BAP and kinetin tested, BAP on WPM was found to be more effective for shoot growth from shoot tip. Under white fluorescent light treatment, shoot growth was much higher than blue, red LED treatments. Root induction from in vitro growth of plantlet was the best on WPM supplemented with IBA. The results suggest that selection of plant growth regulators and light resources could be important factor to achieve an efficient in vitro growth.
An efficient regeneration system was developed for Kigelia pinnata L., a multipurpose tree belonging to the family Bignoniaceae. The nodal segments were cultured in vitro, and the optimum concentrations of plant growth regulators for callus induction were determined. The friable organogenic calli were derived from the basal cut end of the nodal segments The highest yield of morphogenic callus (100%) was observed when nodal segments were cultured on Murashige and Skoog (MS) medium supplemented with 3 μM 2,4 dichlorophenoxyacetic acid (2, 4-D). The morphogenic callus maintained high regeneration during the first four subcultures in the callus induction medium. The maximum shoots (28/culture) were regenerated at the highest frequency of 100% when 3 μM thidiazuron (N-phenyl N′ 1,2,3-thidiazol-5-yl urea) (TDZ) and 0.5 μM naphthaleneacetic acid (NAA) were added to MS medium. The emergence of multiple shoots from the calli was histologically documented. The regenerated shoots showed maximum rooting on 1/2 MS medium containing 4 μM indole-3- butyric acid (IBA). The effect of vesicular arbuscular mycorrhizae (VAM) association in averting the transplantation shock was tested and proved to be highly beneficial, giving a 100% survival rate after 60 d of transplantation. This efficient plant regeneration system provides a foundation for generating transgenic plants of this multipurpose tree.
Rooted hypocotyls from three cultivars of chili pepper (Capsicum annuum were inverted onto Murashige and Skoog (MS) medium with three combinations of benzyladenine (BA) and indoleacetic acid (IAA) in order to induce buds in the exposed cortex. Differences in bud formation were observed for the cultivars tested, with between 4 and 20.1 buds per hypocotyl. The best combination of growth regulator considering all of the cultivars was 5 mg 1−1 of BA and 0.3 mg 1−1 of IAA, since the percentage of bud induction ranged from 46.7 to 100. Hypocotyls bearing buds were placed in the normal position on fresh MS medium, free of growth regulators, allowing the elongation of shoots during a period of 15–30 days. The observed number of elongated shoots per hypocotyl varied from 1.0 to 3.2, depending on the cultivar and the BA/IAA combination. These shoots were excised and rooted on coarse sand, wetted with half strength MS medium.
Tea is one of the most important non-alcoholic beverage drinks worldwide and gaining further popularity as an important health drink. It is served as morning drink for 2/3rd of world population daily. Although conventional breeding and propagation contributed significantly for last several decades for varietal improvement, due to the limitations of conventional breeding coupled with the demand for increasing productivity with lower cost of production, application of biotechnology becomes an alternative approach. Therefore, apart from a dozen of tea research institutes globally, several other groups are working on tea and related genera that have registered many valuable information with several achievements. The present review deals with progress in-depth of various aspects of biotechnological works such as micropropagation and alternative approaches, cell and organ culture techniques, genetic transformation, DNA markers as well as organelle genome and gene cloned from tea and related genera which will be valuable information for the workers working on various aspects of Camellia biotechnology.
Somatic embryos were multiplied by secondary embryogenesis in cotyledonary cultures of peach and nectarine (Prunus persica L.) using a simplified culture medium for immature seeds. A three-stage process with an initial callus phase was established in darkness on a medium containing basal salts (modified MS) supplemented with 2,4-D (5 mg/l), Kn (2 mg/l) and BAP (2 mg/l) and casein hydrolysate (500 mg/l). This was followed by a growth regulator-free medium with activated charcoal for the adventitious and direct multiplication of somatic embryos under continuous light. Somatic embryos (10–15) originated from the epidermal layer of primary somatic embryos of 4–6 mm size. The incidence of morphologically abnormal embryos was reduced by subculturing every 20 days. Calli which were isolated and grown on a 2,4-D medium were more embryogenic than those on NAA. These embryos multiplied continuously for more than 10 months by a repetitive somatic embryogenic process. A third stage medium, supplemented with BAP (2 mg/l), was required for axis elongation, germination and transfer to soil.
A procedure of in vitro plant propagation using shoot meristem explants (∼0.5 cm) has been developed for Capsicum annuum cv CA960, C. baccatum, C. frutescens and C. praetermissum on Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol. 15, 473–497] medium containing various cytokinins. Among various concentrations of cytokinins tested; adenine (Ad), N6-benzyladenine (BA), kinetin (Kn), zeatin and thidiazuron (TDZ) individually. TDZ regenerated maximum number (4.2–22.4) of shoots in all the Capsicum species tested. Multiple shoot elongation occurred upon transfer to BA (0.22 μM l−1) + IAA (0.48 μM l−1). Rooting of regenerated shoots was achieved on medium supplemented with 5.71 μM l−1 indole-acetic acid (IAA). Rooting was observed in 72–94% of shoots obtained from TDZ-containing regeneration medium followed by elongation treatment in contrast to 8–22% of shoots without elongation treatment. Plantlets obtained from TDZ-containing media were normal diploid (2n = 24) and could readily be established in the soil under green house conditions with a survival frequency of 68–84%. Regenerated plants were developed into morphologically normal, fertile plants and able to set viable seeds.
Somaclonal variation in plants regenerated by organogenesis from long-term cultured calluses of two diploid staminate genotypes of Asparagus officinalis cv. Argenteuil was characterized by plant phenotype, ploidy, meiotic behavior, pollen viability, fruit and seed set, and AFLP profiles. Phenotypic deviations from the donors were detected in foliage color, flower size, and cladode and flower morphology. Ploidy changes were observed in 37.8% of the 37 regenerants studied. Meiotic alterations in 12 out of 21 regenerants included laggards, dicentric bridges, micronuclei, restitution nuclei and polyads. Of the 408 AFLP markers screened in 43 regenerants and the donors, 2.94% showed polymorphism. High pollen viability was observed in the 22 regenerants analyzed. All crosses between one pistillate plant and 35 regenerants, as well as the controls, produced fruits and seeds; however, no plump seeds resulted in 35.3% of the crosses with regenerants, and no seeds germinated in 12.5% of those with apparently normal seeds. Fruit and seed set was similar in crosses with diploid regenerants with normal meiosis and the controls but was lower in crosses with diploid and polyploid regenerants with abnormal meiosis. Our results show that the regenerated plants exhibited conspicuous somaclonal variation that could be eventually exploited for in vitro selection systems.
The hepatoprotective effects of the fruits of Hovenia dulcis THUNB. on chemically or immunologically induced experimental liver injury models were examined. The methanol extract showed significant hepatoprotective activity against CCl4-toxicity in rats and D-galactosamine (D-GalN)/lipopolysaccharide-induced liver injury in mice. The methanol extract also significantly protected against CCl4-toxicity in primary cultured rat hepatocytes. Hepatoprotective activity-guided fractionation and chemical analysis led to the isolation of an active constituent, (+)-ampelopsin (1) from the methanol extract.
Over the past fifty years plant breeders have achieved impressive improvements in yield, quality and disease resistance. These gains suggest that many more modifications might be introduced if appropriate genes can be identified. Current DNA techniques allow the construction of transgenic plants and this important new book reviews the current state of knowledge. A team of leading researchers provide in-depth reviews at the cutting edge of technology for laboratory techniques for the transformation of important soil microorganisms and recalcitrant plants of economic value. The book is divided into three sections: soil microorganisms; cereal crops; and industrially important plants. The most effective methods used to date are compared, and their merits and limitations discussed. Some chapters emphasise case studies and applications. In cases where obstacles remain to be overcome, an overview of progress to date is given. The book will serve as a general guide and reference tool for those working on transformation in microbiology and plant science.
Objectives of this study are to make clear the vegetation structure of Hovenia dulcis community in the Korean Peninsula over ten mountains including 17 plots. The results were summarized as follows. Habitat of the community indicated that elevation ranged from 115 meter to 720 meter at the sea level, slope aspect in nearly all directions, bare rock from 0 to 90 percent, slope degree from 10^{\circ} to 40^{\circ}, topography from valley to middle slope, the height of tree layer from 8m to 22m, the diameter at breast height from 12cm to 59cm and coverage from 65% to 95$\%$Vegetation physiognomy was mainly represented by a valley species such as Comus controversa, Quercus serata, Carpinus cordata, Zelkova serrata, Torreya nucifera, Hovenia dulcis, Ulmus davidiana var. japonica, Quercus variabilis, Fagus crenata var. muftinevis, Acer takesimense, etc. Vegetation was largely divided into four types such as Sorbus alnifolia Type, Acer palmatum Type, H. dulcis typical Type and Acer takesimense Type. According to CCA, H. dulcis typical Type was positively correlated with disturbance, bare rock and altitude, S. alnifolia Type indicated a positive correlation with organic matter and A. takesimense Type showed a negative correlation with bare rock. Altitude and slope factors were significantly correlated on axes. Canopy profile of S. alnifolia Type was well developed, H. dulcis typical Type was open with about 55$\%$cover under layer and Acer takesimense Type indicated the lowest canopy height of tree layer In four Types. According to importance value, Hovenia dulcis, Comus kousa, Quercus variabilis, Corpus controversa, Lindera erythrocarpa, etc. indicated high value in the H. dulcis dominated plots, but in other plots, Carpinus cordata, Hovenia dulcis, Torreya nucifera, Quercus serrata, Acer mono, Comus controversa, etc. showed high value caused by the differences of the floristic composition between them.
An efficient regeneration system was developed for Kigelia pinnata L., a multipurpose tree belonging to the family Bignoniaceae. The nodal segments were cultured in vitro, and the optimum concentrations of plant growth regulators for callus induction were determined. The friable organogenic calli were derived from the basal cut end of the nodal segments. The highest yield of morphogenic callus (100%) was observed when nodal segments were cultured on Murashige and Skoog (MS) medium supplemented with 3 muM 2,4 dichlorophenoxyacetic acid (2, 4-D). The morphogenic callus maintained high regeneration during the first four subcultures in the callus induction medium. The maximum shoots (28/culture) were regenerated at the highest frequency of 100% when 3 muM thidiazuron (N-phenyl N' 1,2,3-thidiazol-5-yl urea) (TDZ) and 0.5 muM naphthaleneacetic acid (NAA) were added to MS medium. The emergence of multiple shoots from the calli was histologically documented. The regenerated shoots showed maximum rooting on 1/2 MS medium containing 4 muM indole-3- butyric acid (IBA). The effect of vesicular arbuscular mycorrhizae (VAM) association in averting the transplantation shock was tested and proved to be highly beneficial, giving a 100% survival rate after 60 d of transplantation. This efficient plant regeneration system provides a foundation for generating transgenic plants of this multipurpose tree.
The effect of thidiazuron (TDZ) on the shoot production capacity of preconditioned explants of Castanea sativa Mill. was evaluated. Embryonic axes of chestnut were aseptically germinated for 14 d in Murashige and Skoog (1962) basal medium supplemented with 0.1 mg l -1 TDZ or 1 mg l -1 benzyladenine (BA) (preconditioning medium), and hypocotyl, epicotyl and cotyledonary node explants were then cultured on shoot-induction medium (basal medium containing 0.01 mg l -1 naphthaleneacetic acid in combination with different TDZ concentrations). After four weeks of culture the explants were transferred for a further eight weeks to media with low BA concentrations and no TDZ. Shoots were produced only by cotyledonary node explants which contain preformed meristematic tissue. Best shoot production, in terms of the proportion of explants producing shoots and shoot productivity per explant, was achieved by BA-preconditioned explants exposed to 1-2 mg l -1 TDZ. Fewer TDZ-preconditioned explants produced shoots, and more produced only callus. An histological study showed preconditioned explants to have an altered meristematic axillary region, with meristematic cell proliferation giving rise to expanded, flattened apical meristems and reduced leaf primordium differentiation. Upon shoot-induction treatment the proliferating meristems originated multiple shoot buds without an intervening callus phase. Shoot elongation was achieved by culturing the original explants in Gresshoff and Doy (1972) medium supplemented with 0.05 mg l -1 BA. Shoots longer than 10 mm were subcultured separately to initiate clonal shoot cultures that were proliferated following the outgrowth of axillary shoots. Rooting frequencies greater than 75% were achieved by 16 of the 21 clones evaluated. Rooting capacity was not influenced by either the preconditioning medium or the shoot induction medium, but considerable variation was observed between clones (genotypes).
It was found that the level of alcohol concentration in both mouse and human sera can be significantly decreased up to 42% by oral administration of the mixtures of the extracts of Hovenia dulcis THUNB and Alnus japonica Steud. A single treatment of extract from Hovenia dulcis reduced the serum alcohol concentration to 32%, compared to 13% in treating the extract of Alnus japonica. Similar patterns were observed in enhancing alcohol dehydrogenase (ADH) and glutathion-S-transferase (GST) activity in the liver. The inhibition of cathepsin activity was also greatly reduced by administrating the mixture of both extracts : however, the extract of Alnus japonica did not affect the acitivity of cathepsin. It was concluded that the mixture of both extracts had synergic effect on reducing serum alcohol concentration and improving the detoxification process due to alcohol administration in the liver.
The effects of high-concentration, 2-h liquid pulses of N(6)-benzylaminopurine (BA) and thidiazuron (TD) on adventitious bud and shoot formation were tested in cotyledons of Douglas-fir (Pseudotsuga menziesii). Seedling age proved important; on average, cotyledons from the youngest seedlings formed 10-fold more buds than cotyledons from the oldest seedlings. Optimal cytokinin concentrations for the youngest cotyledons were 400 and 800 μM BA, and 100 and 200 μM TD. Shoots developed best from buds induced with 300, 400, and 800 μM BA. Four gelling agents were tested; BRL agarose yielded more than three times the number of buds, and Gelrite nearly twice the number of buds, as either Sigma agar or Difco Bacto-Agar. One of the best treatments (400 μM BA, agarose) yielded more cotyledons with buds, and more buds per cotyledon, than when cytokinins were incorporated into the growth medium.
In vitro shoot regeneration ability of 17 (7 Italian and 10 Hungarian) bell pepper genotypes was investigated using excised cotyledons and rooted hypocotyls as explants. Most of the Italian genotypes and two of the Hungarian genotypes responded well, producing shoots from rooted hypocotyls. Only two genotypes (one Italian and one Hungarian) gave a weak response using cotyledons. For direct shoot induction in these explants, in addition to the methods cited in the relevant papers, a new method was applied using thidiazuron as a cytokinin. Shoots were successfully regenerated from cotyledons of two Italian and two Hungarian genotypes using thidiazuron which were considered to be non responsive to the usual methods.
Axillary buds of field plants of Cunila galioides Benth. were used to evaluate the effect of growth regulators and culture media on the in vitro shoot proliferation and growing. The highest multiplication rate was obtained using Murashige and Skoog (MS) medium supplemented with 8.8 M of benzyladenine. Repeated subcultures of shoot tips and single nodes at 4-week intervals for eight months on the above medium enabled mass multiplication of shoots without any evidence of decline. The best conditions for rooting were MS medium plus 0.5 to 2.5 M of indolebutyric acid. The rooted plants were successfully transferred to soil, exhibiting a normal development.
The molecular mechanisms that regulate the transcription of key developmental genes involved in shoot organogenesis have yet to be fully elucidated. However, it is clear that plant growth regulators, such as cytokinin, play a critical role in the differentiation of adventitious shoots. In Nicotiana tabacum zz100 leaf discs, high frequency shoot formation could be induced with 5 μM of the cytokinin N
6-benzyladenine (BA). Increasing the exogenous BA concentration to greater than 20 μM resulted in stunted explants with abnormal shoot morphology and altered mineral composition. Explants with abnormal shoots did not appear to be hyperhydric. Abnormalities were, however, associated with an increase in the expression of a knotted1-type homeobox gene (TobH1) isolated from normal shoot-forming cultures. The results suggest that the development of cytokinin-induced abnormal shoot morphology possibly involves changes in TobH1 gene expression.
Many available methodologies for in vitro regeneration of commercial tomato varieties promote not only the production of normal shoots but also individual leaves, shoots without apical meristems and vitrified structures. All these abnormal formations influence and diminish the regeneration efficiency. At the basis of this phenomenon lies callus development. We optimized an alternative procedure by which the regeneration occurs without abnormal shoot formation. The portion including the proximal part of hypocotyls and the radicle was cultured on medium consisting of Murashige and Skoog salts, 4 mg/L thiamine, 100 mg/L mio-inositol and 3% sucrose. After two-three weeks, 60% explants showed adventitious shoot formation. No changes in the morphological characteristics of regenerated plants and fruits were observed as compared with parents. Karyotypic analysis of regenerated plants showed no variations in chromosome number. The optimized procedure offers the advantage of tomato plant regeneration avoiding callus formation, which enables normal plant recovery with an efficiency ranging from 1.45 +/- 0.05 to 2.57 +/- 0.06 shoots per explant in Campbell-28, Amalia, Lignon, and Floradel cultivars.
Somatic embryogenesis and plant regeneration of Hovenia dulcis Thunb
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