Organogenic Response Research Articles

Thin Cell Layer Tissue Culture Technology with Emphasis on Tree Species

An increased dependency on plant-based resources for food, shelter, and medicinal usage has increased their sustainable and unsustainable exploitation. To use this resource sustainably, plant tissue culture (PTC) is one important technology. Among different PTC techniques, thin cell layer (TCL) technology is a relatively simple and easily adaptable technique for in vitro cultures of plants. This technique uses small explants about 0.5–2 mm in thickness excised from different plant organs. It has been successfully used in the large-scale propagation of vegetables, legumes, and plants with medicinal benefits. TCL technology has proven to be effective in stimulating various organogenic responses when combined with various new methods such as nanotechnology or microtome-based explantation, especially in tree species. It is considered an important tool in plant biotechnology. Although the morphogenetic response per explant is usually higher in conventional explants, the appropriate use of plant growth regulators and geometric factors in TCL has the potential to make it more efficient and beneficial. This article provides an overview of the concept of TCL as applied to different plant species, particularly trees, since there are few, if any, summaries of TCL technology, especially in trees. This review will certainly revitalize this important technology so that it can be used effectively for successful mass propagation in the field of plant tissue culture.

In Vitro Growth and Regeneration of Brassica oleracea var. gongylodes: A Decade of Research

Kohlrabi (Brassica oleracea var. gongylodes) is a vegetable cultivated for its edible stem tuber. Although valued for its nutritional properties and tolerance to abiotic stress, kohlrabi is one of the least studied brassicas. In this review, we summarize the results of our decade-long research on in vitro morphogenesis of kohlrabi, starting from 2013. Protocols for efficient in vitro regeneration with minimal requirements for external application of plant growth regulators (PGRs) have been developed, both by somatic embryogenesis (SE) and by de novo shoot organogenesis (DNSO). Efficient regeneration by direct SE was achieved using immature zygotic embryos as explants incubated on PGR-free media, and the production process was maintained in culture thanks to highly efficient secondary SE. Conversely, efficient regeneration by indirect DNSO was achieved using entire seedlings as explants and adding only cytokinin (CK) without the need for exogenous auxin. Comprehensive phytohormone analyses revealed that different exogenously applied CKs differentially affected the composition of endogenous phytohormones and induced changes in the expression of cell cycle-related genes and other genes involved in the organogenic response. The addition of high sucrose concentrations to the nutrient media failed to induce the formation of stem tubers in in vitro culture, but revealed a complex interaction with exogenously applied CKs, interfering with both the endogenous phytohormonome and the expression of organogenesis-related genes. Our work has provided substantial biotechnological advances in the field of in vitro regeneration of kohlrabi, as well as in understanding the underlying phytohormonal regulation. The review aims to introduce kohlrabi to the scientific community as a model system for both basic and applied research, while we continue seeking answers to the outstanding questions and trying to pave the way for the development of more resistant varieties.

Acceleration of Callus Formation in Date Palm (Phoenix dactylifera L.) Zygotic Embryos

This study was carried out to test the effect of water soaking ofseeds of date palm ( P. dactylifera L.) as a pre-empryo excision treatment,on in vitro culture of zygotic empryos. The objective was optimization oforganogenic callus formation. Mature seeds of “Barakawi” and “MishrigWad Laggi” date palm cultivars were used as sources of embryos.Durations of 0- up to 9-days of seed water soaking, prior to embryoexcision and culture, were tested. Murashige and Skoog (1962) mediumwas used as basal medium. The cultures responded positively to the watersoaking treatments and resulted in the organogenic responses of swelling,germination, callus and adventitious roots formation were obtained. Themagnitude and type of response varied with cultivar and soaking duration.All water soaking treatments increased the measured responses relative tothe control. The results showed that a 6-day soaking treatment wasoptimal for all measured responses compared with the other periods ofsoaking tested. Calli formation in 6-day water soaking treatment were50% and 70% for “Barakawi” and “Mishrig Wad Laggai” ,respectively.However, increasing the soaking duration beyond the 6-day had a markednegative effect on morphogenic response regardless of cultivar. The bestresults for all measured parameters were obtained with embryos of“Mishrig Wad Laggi” compared with embryos excised from “Barakawi”.It seems that soaking date palm seeds in water prior to embryo excisionplay an important role, in enhancing morphogenic response and callusformation in date palm tissue culture.

Effect of Nitrogen on in Vitro Propagation of Endangered Medicinal Plant: Swertia Chirayita Roxb. Ex Flaming

The effect of nitrogen was investigated on the organogenesis of Swertia chirayita (Gentianaceae) to overcome the challenges related to its cultivation. The best callogenic response was observed on root explants inoculated onto MS medium supplemented with BAP (2.0 mg/l) along with 2,4-D (0.5 mg/l) after 35 days of culture. Subsequent transfer of callus for multiplication on the same media composition under complete darkness presents the best results in terms of callus multiplication. Callogenic cultures were subculture onto modified MS medium supplemented with inorganic nitrogen sources, i.e., NH4NO3 (14-56N/l), KNO3 (100-400N/l) with BAP (3.0 mg/l) were observed. Organogenic response (52%) was observed after 8-12 weeks of culturing. The maximum number of the shoot was recorded on MS medium with NH4NO3 (28 N/l), KNO3 (300N/l) with BAP (3.0 mg/l). Moreover, 90% of them were able to regrow when sub-cultured on the same media. Sixteen weeks old multiple shoots were subcultured on MS medium supplemented with different auxins. IAA was proved to be the best hormone rooting purpose. However, the best rooting response regarding the number of roots and an average length of roots was obtained at IAA (1.0 mg/l). Survival of 90% was achieved when rooted plantlets were successfully established in substrate containing sand, vermicompost, and garden soil in equal proportion for hardening and acclimatized.

Effect of medium composition, genotype and age of explant on the regeneration of hexaploid plants from endosperm culture of tetraploid kiwiberry (Actinidia arguta)

Endosperm, an ephemeral and storage tissue, serves as a source of nutrition and protection during embryo development and germination. It can be used for the cultivation of polyploid plants in vitro. Here, results of plant regeneration and acclimatization from the endosperm-derived calli of four cultivars of Actinidia arguta has been presented. Seeds excised from fresh fruit and dry seeds stored for one year served as the sources of endosperm explants of selected tetraploid cultivars of A. arguta. Callus Induction Medium (CIM; containing 0.25, 0.5, or 1 mg/l of TDZ) and Actinidia Endosperm Medium (AEM; containing 2 mg/l of 2,4-D and 5 mg/l of kinetin) were used to study the organogenic responses of the calli. On AEM, the source of explant did not significantly affect the rate of callus induction for any of the tested cultivars; no organogenic events were observed. In contrast, on CIM both the source of explants and the cultivar origin caused significant differences in callus formation and subsequent organogenic events. Histological and ultrastructural analyses revealed the adventitious nature of shoot bud formation on these media. The most efficient elongation of shoot buds was achieved after transferring organogenic calli with adventitious shoot buds to a medium supplemented with zeatin or meta-topolin. Robust root induction with minimal basal callus formation occurred on the medium with indole-3-acetic acid. Flow cytometric analysis revealed that the nuclear DNA content in the leaves of some regenerants was approximately 50 % higher (4.5 pg/2C) than that in leaves from the tetraploid seedlings (3.1 pg/2C),which confirmed that those regenerants originated from the endosperm. The regeneration of such hexaploid plants was more efficient when endosperm from fresh seeds served as an explant; therefore, fresh rather than dry seeds are recommended for endosperm-derived plant production. The hexaploid plants of A. arguta can serve as an important source of breeding material.

In vitro propagation of Nanorrhinum ramosissimum (Wall.) Betsche: A traditionally important medicinal plant

An efficient micropropagation protocol facilitates successful conservation and improvement of Nanorrhinum ramosissimum (Wall.) Betsche by biotechnological means. Shoot tip explants exhibited optimal organogenic response when inoculated on half-strength(1/2) Murashige and Skoog (MS) medium supplemented with kinetin (KN) and indole-3-acetic acid (IAA) (0.5 mg/L each). Shoot organogenesis was further enhanced when the multiplication medium was fortified with dextrose (1%) (2.6 shoots/explant; 7.9 cm shoot length). The regenerated shoots formed roots; however, the best rooting frequency (87%) was achieved on half-strength MS medium containing only IAA (0.5 mg/L). Four-week-old in vitro plantlets were acclimatized with 95% survival under greenhouse conditions. The regeneration protocol developed in this study can be utilized for germplasm conservation of this elite traditional medicinal plant.

Organogenesis from Leaf Tissue of Spondias pinnata (L. f.) Kurz, SEM study and Genetic Fidelity Assessment by ISSR and ScoT

In vitro raised plantlets were obtained from nodal tissue through direct organogenesis and they served as donor plants for the collection of leaf explants. Leaf explants were inoculated on Murashige and Skoog’s medium with different concentrations of 2,4-Dichlorophenoxyacetic acid (2,4-D). Hundred percent callogenesis was observed on medium supplemented with 5 mg l−1 2,4-D. For the multiplication of cells (proliferation), calli were transferred to either basal medium or media containing different types and concentrations of cytokinins. Proliferation was observed maximum on media containing 0.5 mg l−1 BAP or 1.0 mg l−1 BAP. Shoot differentiation from calli took place on media supplemented with BAP in combinations with TDZ or ZN. Initiation of organogenesis was observed in calli within two weeks of their subculture on differentiation medium. Shoot differentiation was maximum, when calli proliferated on medium having 1 mg l−1 BAP were transferred to the medium containing 1 mg l−1 BAP and 0.5 mg l−1 TDZ. Organogenic responses after four weeks of subculture on above differentiation medium were as such: number of shoots per explants (25.33 ± 0.88), number of shoots per calli replicate (3.67 ± 0.33) and maximum shoot length (3.43 ± 0.20 cm). Medium supplemented with 2.5 mg l−1 NAA was most responsive for rooting of shoots (54.16 ± 1.39%). About 62.5% plantlets survived after hardening and 54.17% plantlets got acclimatized. All acclimatized plants were transferred to field condition successfully. Formation of unipolar shoots and their multicellular attachment with callus were observed by scanning electron microscopy. To confirm the genetic fidelity of micropropagated plants, five micropropagated plants derived from different leaf explants and two mother plants (randomly selected from micropropagated plants raised from nodal explants) were subjected to molecular analysis. The genetic fidelity of in vitro regenerated plants was assessed by using SCoT and ISSR molecular markers. 12.5% polymorphism was reported in both studies, which may be due to callus mediated regeneration of shoots. Indirect organogenesis from leaf tissues of Spondias pinnata can be used both for conservation as well as the improvement of plants.

Hormonal Regulation for <i>de novo</i> Organogenesis in <i>in vtro</i> Derived Leaf and Internode of <i>dAegle marmelos</i> L.

Adventitious shoot formation from internode explants of Aegle marmelos was achieved. Leaf and internode explants were procured from shoots obtained from in vitro germinating seedlings. Callogenesis was induced from leaf and internode explants on Murashige and Skoog (MS) semisolid medium variously supplemented with 6-Benzyladenine (BA) and 2, 4-Dicholophenoxyacetic acid (2, 4-D) while the organogenic response was found remarkable only in BA supplemented medium. Both types of explant markedly influenced callus formation while organogenesis was noticed only in internode explants. The number of shoots formed in internode explants was greater in the medium supplemented with 0.5µM BA and registered 6.86 numbers of shoot. The regenerated shoots transferred to half MS medium supplemented with 2mg/l IBA produced 75% rooting followed by 66% in IAA supplemented medium. The plantlets raised in vitro were successfully hardened to ex vitro conditions.

Evaluation of subculture ages on organogenic response from root callus and SPAR based genetic fidelity assessment in the regenerants of Hibiscus sabdariffa L.

The efficacy of an organogenesis based regeneration protocol and the occurrence of somaclonal variation has been studied up-to eighth subculture of Hibiscus sabdariffa L. var. HS4288. Initially, the induction of organogenic calli from 83.33% root explants was standardized in 2.26 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65 μM kinetin (KIN) supplemented Driver-Kuniyuki Walnut (DKW) basal medium. The calli were maintained up to the eighth subculture (each with 6-weeks duration) in the same basal medium along with 1.13 μM 2, 4-D and 4.65 μM KIN. The highest regenerative efficiency of the root-derived calli was found in 1.08 μM α-Naphthaleneacetic acid (NAA) and 8.88 μM 6-Bezyladenine (BA) containing DKW medium. This particular medium produced 14.30 ± 0.47 numbers of visible shoots (>10 mm in length) at the first subculture from 425 ± 35 mg of callus through the process of organogenesis. The morpho-histological study confirmed the organogenic mode of regeneration. With the gradual increase of culture passage, the frequency of shoots was steadily declined. The genetic polymorphism of the regenerated plants from eight successive sub-cultures was assessed using three different single primer amplification reaction (SPAR) methods; viz. Random amplified polymorphic DNA (RAPD), Direct amplification of minisatellite DNA (DAMD) and Inter simple sequence repeat (ISSR) markers. The SPAR analysis revealed that up to fifth subculture all the three types of primers produced the monomorphic banding pattern among the regenerated plants with that of their mother plant. From the sixth subculture onwards there were some alterations in banding profiles both in RAPD and ISSR markers but DAMD marker did not show any polymorphism up to eighth subculture. In the present work, the highest polymorphism percentage was recorded among the plants of eighth subculture by using both RAPD (41.61%) and ISSR (31.65%) markers. The SPAR method ensured that the present protocol may be used for true to type plant production up-to five consecutive subcultures.

Morphogenesis and in vitro production of caffeoylquinic and caffeic acids in Baccharis conferta Kunth

We established a protocol for the in vitro propagation of Baccharis conferta Kunth. This plant is used to treat gastrointestinal problems, cramps, pain, respiratory problems, and insect bites. A high rate of shoot multiplication was obtained from nodal segments on Murashige and Skoog (MS) culture medium. The shoots regenerated roots without exogenous plant growth regulators (PGRs). All explants of wild leaves on MS medium containing 5 μM of thidiazuron (TDZ) produced friable callus. An organogenic response was achieved after 3 wk of culture when callus segments were transferred to MS medium containing a combination of plant growth regulators (PGRs): either (i) 5 μM indole butyric acid (IBA) + 5 μM kinetin (KIN) or (ii) 0.5 μM IBA + 1.10 μM benzylaminopurine (BAP). The morphogenetic responses of callus were characterized by scanning electron microscopy. Shoots regenerated from callus and formed roots on MS medium without PGRs. The micropropagated plantlets and the organogenic callus showed similar chemical profiles in HPLC-mass spectrometry analyses. The main compounds present in the cultures were caffeoylquinic acids. Only plantlets contained small amounts of triterpenes (erythrodiol and ursolic acid). These findings will be useful for the micropropagation of this important native resource, and for further studies on its biology.

Wounding and medium formulation affect de novo shoot organogenic responses in hypocotyl-derived explants of annatto (Bixa orellana L.)

Bixa orellana L. (annatto) is the unique exploited source for the production of bixin and norbixin natural dyes, which are widely used in the food, pharmaceutical, cosmetic, and textile industries. In tissue culture, adequate culture conditions are essential for the development of the plant. Among them, the saline composition of the culture medium and the type of explants are limiting factors. In this study, the effect of MS (Murashige and Skoog), Correia and colleagues (JADS) medium, Woody Plant Medium (WPM), and Driver Kuniyuki Walnut (DKW) culture media and wounding of hypocotyl explants on de novo shoot organogenesis (DNSO) of annatto were evaluated. It was demonstrated that JADS medium improved the nutritional status of shoots, which promoted the best development and quality of the plants generated after acclimatization, when compared to MS medium. The use of JADS medium for annatto DNSO during 45 d of culture was sufficient to regenerate and stimulate the growth of shoots, which had adequate elongation and rhizogenic responses. In addition, enlargement of the wound surface, using longitudinal sections of the hypocotyl explant cut surface down on JADS medium, significantly improved the frequency and number of newly formed shoots. This protocol enabled production of an average of 17 30-mm-long shoots per explant after 90 d of culture. These results will be useful for future studies related to annatto biotechnology.

Deciphering the growth pattern and phytohormonal content in Saskatoon berry (Amelanchier alnifolia) in response to in vitro cytokinin application

Clonal propagation plays a critical integral role in the growth and success of a global multi-billion dollar horticulture industry through a constant supply of healthy stock plants. The supply chain depends on continuously improving the micropropagation process, thus, understanding the physiology of in vitro plants remains a core component. We evaluated the influence of exogenously applied cytokinins (CKs, N6-benzyladenine = BA, isopentenyladenine = iP, meta-topolin = mT, 6-(3-hydroxybenzylamino)-9-(tetrahydropyran-2-yl)purine = mTTHP) in Murashige and Skoog (MS)-supplemented media on organogenic response and accumulation of endogenous CK and indole-3-acetic acid (IAA) metabolites. The highest shoot proliferation (30 shoots/explant) was obtained with 20 μM mT treatment. However, the best quality regenerants were produced in 10 μM mT treatment. Rooting of Amelanchier alnifolia in vitro plantlets was observed at the lowest CK concentrations, with the highest root proliferation (3 roots/explant) in 1 μM mTTHP regenerants. Similar to the organogenic response, high levels of endogenous bioactive CK metabolites (free bases, ribosides, and nucleotides) were detected in mT and mTTHP-derived regenerants. The level of O-glucosides was also comparatively high in these cultures. All CK-treated plants had high levels of endogenous free IAA compared to the control. This may suggest an influence of CKs on biosynthesis of IAA.

In vitro formation of adventitious shoots on caulinary tissue of physiologically contrasting Agave angustifolia plants

To micropropagate agave plants, somatic tissue is obtained from selected plants that are conditioned for 2 to 6 months to improve their physiological condition and health before in vitro cultivation. The objective of this study was to evaluate the physiological condition of Agave angustifolia plants in terms of its effect on organogenic response in somatic tissue taken from these plants when they are established in similar culture media. In a nursery, growth of four groups of plants was evaluated when they were subjected to different types of irrigation for seven months: 1) water; 2) NS-50% (fertigation with nutrient solution at 50% strength); 3) NS-75%; and 4) NS-100%. At the end of the period, it was found that supplying nutrients is important for plants to achieve better physiological condition. The unfertilized plants and those that received NS-75% had increases of 3.8 and 7.8 leaves, 6.5 and 12.5 cm in length of the largest leaf, and 1610.3 and 4401.4 cm2 in leaf area. Stem tissue was obtained from these stock plants and cultured for 90 days in in vitro culture, and formation of adventitious shoots was assessed. The results showed that the magnitude of organogenesis in stem tissue for formation of adventitious shoots was positively related to the physiological condition of the stock plant. Explants taken from unfertilized stock plants formed 14.6 total shoots and 3.8 shoots on each explant, while those fertigated at 100% concentration of nutrients formed 32.7 total shoots and 8 shoots on each explant.

In vitro multiplication and genetic stability of two species of Micranthocereus Backeb. (Cactaceae) endemic to Bahia, Brazil

The two targeted species of this study, Micranthocereus flaviflorus subsp. densiflorus and M. polyanthus subsp. alvinii, are endemic to the state of Bahia and have ornamental value. The main goal of this work was to micropropagate these species and to evaluate the genetic stability of the regenerated plants. To do so, shoots originated from in vitro germinated plants were inoculated in MS/2 (Murashige, Skoog, Physiol Plant 15:473–497, 1962) media containing 1.34 μmol L−1 of α-naphthaleneacetic acid (NAA) for morphogenesis induction. This was repeated for three consecutive subcultures, and the subcultured shoots were designated by order of production as S1, S2 and S3. Retention of morphogenic potential and acceleration of organogenic response was observed after the three subsequent propagation events, so that if shoots were used as explant source the in vitro propagation of M. flaviflorus could be achieved in 90 days and that of M. polyanthus could be optimized to 60 days of duration. In order to perform genetic stability analysis along subcultures, ISSR markers were used and genetic variation between shoots of each subculture and their donor plant was measured with Jaccard’s similarity coefficient. This analysis revealed high genetic stability in the in vitro propagation of all the donor plants of M. flaviflorus and M. polyanthus in regards to three consecutive shoot subcultures, in which similarities were 100% for both species. The study of a greater number of subcultures is suggested to assess morphogenesis potential and genetic fidelity in long term.

Comparative Assessment of Response to Cadmium in Heavy Metal-Tolerant Shrubs Cultured In Vitro

Two species of Pb-adapted shrubs, Alyssum montanum and Daphne jasminea, were evaluated in vitro for their tolerance to elevated concentrations of cadmium. Shoot cultures were treated with 0.5, 2.5, and 5.0 μM CdCl2 for 16 weeks and analyzed for their organogenic response, biomass accretion, pigment content, and macronutrient status. Cadmium accumulation and its root-to-shoot translocation were also determined. In both species, rooted microplantlets, suitable for acclimatization, were obtained in the presence of Cd applied as selection agent. In A. montanum, low and moderate dose of Cd stimulated multiplication, rooting, and biomass production. Growth tolerance index (GTI) in Cd-treated shoots ranged from 120 to 215%, while in the roots 51–202%. In turn, in Cd-treated D. jasminea proliferation and rooting were inhibited, and GTI for shoots decreased with increasing doses of Cd. However, roots exposed to Cd had higher biomass accretion. Both species accumulated Cd in developed organs, and its content increased with increasing CdCl2 dose. Interestingly, D. jasminea accumulated higher amounts of Cd in the roots than A. montanum and immobilized this metal in the root system. On the contrary, A. montanum translocated some part of accumulated Cd to the shoots, but with low efficiency. In the presence of Cd, A. montanum maintained macronutrient homeostasis and synthesized higher amounts of phytosynthetic pigments in the shoots. D. jasminea accumulated root biomass, immobilized Cd, and restricted its translocation at the expense of nutrient balance. Considering remediation potential, A. montanum could be exploited in phytoextraction, while D. jasminea in phytostabilization of polluted substrate.

Induction of Somatic Embryogenesis from Cocoa Farmer Field Collection of ICT - Peru

To induce the formation of embryogenic structures from staminodes, in 10 cocoa genotypes (Theobroma cacao L.) from the ICT Collection, the influence of the combination of two growth hormones (2,4 Dichlorophenoxyacetic 2,4 -D and Thidiazuron TDZ) was studied. Floral explants (staminodes), were induced to form calli in a culture medium supplemented with modified DKW mineral salts. At 12 days of implantation in the culture medium, the explants responded to the callus induction with a bulge at the base, covering the explants totally with callus at 40 days of culture. Embryogenic expression was shown at 52 days of culture. The genotypes ICT-2161 and ICT-2142 shows the highest percentage of somatic embryogenesis. Four types of callus, fine crystalline, thick crystalline, semi-compact and compact were observed. In addition, a clear organogenic response was observed when roots were formed from the callus generated.

Influence of Meta-Topolin on Efficient Plant Regeneration via Micropropagation and Organogenesis of Safflower (<i>Carthamus tinctorius</i> L.) cv. NARI-H-15

The effect of meta-Topolin (mT) was assessed to develop a reliable protocol for efficient plant regeneration of safflower (Carthamus tinctorius L.) cv. NARI-H-15. For micropropagation, 7 - 9 days old shoot-tip explants cultured on MS basal medium supplemented with 3.0 mg/L meta-Topolin (mT) + 0.5 mg/L CPPU showed 97.7% adventitious shoot formation (42.4 shootlets) than node after 45 days of culture. For organogenesis, the seedling explants of immature leaf cultured on 1.5 mg/L CPPU or 1.5 mg/L NAA fortified medium produced high amount of callus than cotyledon and stem calli after 60 days of culture. However, MS basal medium fortified with 4.0 mg/L mT + 1.5 mg/L CPPU was found beneficial to stimulate 100% organogenic response (74.7 shootlets) from immature leaf calli than cotyledon and stem derived calli after 45 days of culture. The healthy plantlets obtained from micropropagation and organogenesis process cultured on 1/4 MS basal salts, 1.5% sucrose (w/v) and 0.8% agar (w/v) medium supplemented with NAA (1.5 mg/L) and mT (0.1 mg/L) produced maximum of 96% (12.8 rootlets) and 84% (7.3 rootlets) adventitious rooting, respectively than mT and CPPU tested medium. However, maximum of 67% and 42% survival rate was noticed when in vitro raised plants from micropropagation and organogenesis were hardened in pots containing soil mix and maintained under green house condition. This optimized regeneration protocol might be helpful in regeneration of new genotypes and cultivars of safflower to improve agronomic traits through in vitro selection process and Agrobacterium-mediated genetic transformation system.

Adventitious shoot organogenesis from leaf and petiole explants of European hazelnut

Adventitious shoot organogenesis and somatic embryogenesis are the basis for implementing new genetic variability and biotechnological approaches in woody species, particularly if mature tissues from valuable cultivars are used. To date, these technologies are only applied to few tree species due to the absence of efficient regeneration protocols. In hazelnut, adventitious shoot organogenesis and somatic embryogenesis have only been carried out with zygotic embryo tissues up to now. Here we report plant regeneration from explants of somatic origin by using in vitro rejuvenated mature tissues (leaves, petioles and stipules). A histological analysis carried out on calli grown on various media, showed significant evidence of shoot regeneration, as proved by the presence of vascular elements such as tracheids with annular or helical secondary wall thickening. Subsequently, the optimization of the regeneration protocol performed by pre-treating the explants with antibiotics (carbenicillin, vancomycin, and cefotaxime) as molecules with auxin-like effects enabled us to achieve shoot organogenesis in hazelnut. The organogenic competence strictly depended on the explants and antibiotics used in the experiments. Following an antibiotic pre-treatment of cv Tonda Gentile Romana explants in the proliferation stage, organogenic responses (frequencies of 40 %) were obtained. According to the results obtained, the best protocol for inducing shoot organogenesis in hazelnut should include the use of explants (leaves and petioles) conditioned in a double-liquid layer of cefotaxime 1000 mg L−1 added to the proliferation medium, cultured on the induction medium, consisting in solid MS medium supplemented with 3 % sucrose, 6-benzylaminopurine 1 mg L−1, indole-3-butyric acid 1 mg L−1 and kinetin 2 mg L−1, and then by sub-culturing the newly-formed calli to regeneration medium, consisting of half-strength solid MS medium with sucrose (30 g L−1) and 6-benzylaminopurine (0.5 mg L−1).

Direct Organogenesis of Stevia rebaudiana Bertoni Using Thin Cell Layer (TCL) Method

An effective protocol was established for the micropropagation of Stevia rebaudiana Bertoni (Asteraceae) through direct organogenesis using thin cell layer (TCL) method. Different concentrations (0, 2.22, 4.44, and 6.66 µM) of exogenous growth regulators [Benzyladenine (BA) and (0, 2.26, 4.52, and 6.78 µM) of 2,4-dichlorophenoxyacetic acid (2,4-D)] were evaluated in combination with transverse thin cell layers (tTCLs) of the hypocotyls of seedlings established in vitro in MS medium. The best organogenic response (31.16 shoots per tTCL explant) was obtained by applying 6.78 µM of 2,4-D. The effect of incubation conditions on the morphogenetic responses was evaluated. Combination of conditions of photoperiod and complete darkness presented the best results in terms of the appearance of shoots vigor. Roots formed on 100 % of the shoots on using ½ MS without PGRs. Of the regenerated plants, 90 % acclimated successfully under greenhouse conditions.

Respuestas morfogénicas de tres explantes de Lupinus montanus (hbk) cultivados in vitro

El necrosamiento y la respuesta morfogénica de explantes de cotiledón, epicótilo e hipocótilo de plántulas de Lupinus montanus (HBK) germinadas in vitro se evaluaron para establecer las condiciones básicas de micropropagación. El necrosamiento se evaluó en medio MS con 0.40 mg·litro-1 de tiamina, 100 mg·litro-1 de mio-inositol, 3 % de sacarosa y 7 g·litro-1 de agar-agar y distintas dosis de carbón activado (CA) y ácido cítrico. Los tratamientos con CA (50 mg·litro-1 y 100 mg·litro-1) fueron los mejores en el control del necrosamiento (P = 0.001); el cotiledón tuvo mayor nivel de necrosamiento (< 60 %) que el epicótilo e hipocótilo (entre 10 y 30 %). Las respuestas morfogénicas se analizaron en medio MS con 0.40 mg·litro-1 de tiamina, 100 mg·litro-1 de mio-inositol, 3 % de sacarosa, 7 g·litro-1 de agar-agar, 100 mg·litro-1 de CA y cinco combinaciones de AIA (ácido indol acético) y BA (6-benciladenina). Los explantes de hipocótilo y epicótilo mostraron mayor respuesta organogénica (> 70 %) a diferencia del cotiledón, cuya respuesta fue primordialmente callogénica (50 %). El epicótilo cultivado en medio MS con 3.0 μM de AIA y 1.0 μM de BA tuvo el mayor número de brotes (10) y altura (11.4 ± 2.6 cm).