Number Of Adventitious Shoots Research Articles

In vitro shoot regeneration system from leaves wrapped by bud scales of a multipurpose tree (Neolamarckia cadamba)

Neolamarckia cadamba (N. cadamba) is an evergreen tree species known for its rapid growth, remarkable wood properties, and significant value in medicine, feeding, and landscape. In order to clone a N. cadamba individual with excellent genotype, a plant regeneration protocol was successfully established with leaves wrapped by bud scales as explants. The optimal sterilization method for the leaves was 0.1% Mercury Chloride (HgCl2) treatment for 1 min before culturing on Murashige and Skoog’s medium (MS) supplemented with 3.0 mg/L Thidiazuron (TDZ), 0.1 mg/L 2–4 Dichlorophenoxyacetic acid (2-4D), 0.05 mg/L α-Naphthaleneacetic acid (NAA) and 1 mL/L Plant Preservative Mixture (PPM) to induce calluses. The medium containing 1 mL/L PPM could effectively inhibit explant contamination without an unfavorable impact on the final induction rate of callus from the leaves. Three types of calluses were induced from the leaves cultured on the above medium. Among them, only the Type II callus, which was green and nodular, had few particle masses, could differentiate into adventitious shoots on the MS medium supplemented with 1.5 mg/L 6–Benzylaminopurine (6-BA) and 0.05 mg/L NAA, with the induction rate of 78.89% and adventitious shoot number per callus of 11.67. The adventitious shoots were proliferated on the MS medium supplemented with 1.0 mg/L 6-BA and 0.05 mg/L Indole-3- butyric acid (IBA) with the proliferation coefficient of 3.37. And the micro-shoots developed roots in the MS medium supplemented with 0.05 mg/L NAA and 0.05 mg/L IBA. The regeneration protocol can be used in the propagation and large scale production of seedlings with the same genotype as an excellent individual of N. cadamba in the field.

In Vitro Conservation of Prunus Avium, an Endangered Species in Tunisia, Using a Plant Extract of Rubus Fruticosus Compared to Indole-3-Butyric Acid

Prunus avium L. is the rarest forest fruit tree in northwestern Tunisia, with strong socio-economic, agronomic, and commercial potential. The aim of this study was to provide more information about the domestication of this species by using an inexpensive in vitro culture technique for micropropagation of meristems. The plant extract used in this study was Rubus fruticosus. The results showed that the best concentration of the extract of R. fruticosus applied for the budding of meristematic microcuttings was 15 g/L, with a 100% success rate. The maximum number of adventitious shoots (7.15 ± 0.35) was obtained for the meristematic microcuttings at a concentration of 15 g/L of the extract. These results indicated that the extracted R. fruticosus plant promotes the neoformation of adventitious shoots and leaves. Rhizogenesis was strongly favored by the addition of 1 mg/L of IBA and 15 g/L of plant extract to the MS medium, which leads to the formation of roots for the meristematic micro-cuttings with percentages of about 80.36% and 53.32%, respectively. The current investigation proved that in vitro micropropagation could constitute a good alternative to the regeneration of this rare forest fruit species and may be applicable to other endangered plants.

In Vitro Regeneration, Conservation, and Field Evaluation of a Medicinal Plant– Greater Burdock (Arctium Lappa L.)

A suitable micropropagation protocol and ex vitro acclimation method have been developed from in vitro grown seedling explants through cotyledonary node and leaf explants in consideration of the vegetable and medicinal properties of Greater Burdock. MS medium with 0.5-4.0 μM BAP showed highest percentage of axillary shoot regeneration from the cotyledonary nodal explants. Direct shoot regeneration was achieved by culturing 1.0 cm2 sections of about 25 days old leaves of in vitro grown shoot on MS medium enriched with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. Within six weeks of incubation on medium enriched with 4.0 M BAP and 2.0 M IBA or NAA, the leaf explants also developed callus from the cut margins. The greatest number of adventitious shoots could then be formed from the leaf-derived callus within 10 weeks of culture on the same media mix. More than 20 shoots were formed per callus clump at the third subculture, which had the highest rate of shoot multiplication. A. lappa's shoot and callus were both preserved at 5 ºC in MS medium with 4.0 μM Kn and 2.0 μM IBA, as well as 4.0 μM BAP and 2.0 μM IBA, respectively. The in vitro proliferated and elongated shoots were separated from callus clump for rooting. A root-induction MS medium with 6.0 μM IBA or NAA was used to cultivate the microshoots individually. All of the cultured microshoots generated 2-16 roots within 4 weeks of being moved to the rooting medium. Regenerated plantlets were transferred to vermiculite and successfully established in an ex vivo environment with a 98% survival rate. J. Bio-Sci. 31(1): 1-15, 2023

Optimization of Callus Induction and Shoot Regeneration from Tomato Cotyledon Explants.

Cultivated tomato (Solanum lycopersicum L.) is one of the most important horticultural crops in the world. The optimization of culture media for callus formation and tissue regeneration of different tomato genotypes presents numerous biotechnological applications. In this work, we have analyzed the effect of different concentrations of zeatin and indole-3-acetic acid on the regeneration of cotyledon explants in tomato cultivars M82 and Micro-Tom. We evaluated regeneration parameters such as the percentage of callus formation and the area of callus formed, as well as the initiation percentage and the number of adventitious shoots. The best hormone combination produced shoot-like structures after 2-3 weeks. We observed the formation of leaf primordia from these structures after about 3-4 weeks. Upon transferring the regenerating micro-stems to a defined growth medium, it was possible to obtain whole plantlets between 4 and 6 weeks. This hormone combination was applied to other genotypes of S. lycopersicum, including commercial varieties and ancestral tomato varieties. Our method is suitable for obtaining many plantlets of different tomato genotypes from cotyledon explants in a very short time, with direct applications for plant transformation, use of gene editing techniques, and vegetative propagation of elite cultivars.

<i>In Vitro</i> Callus Induction and Adventitious Shoot Regeneration from Leaf Explants of Heuchera Hybrid

The problems of morphogenesis’ induction from isolated leaf explants of the variety Georgia Plum of Heuchera hybrid were studied for the first time. The induction of callus and of adventitious shoots from leaf explants on the modified MS medium containing 30g/l glucose was carried out in the presence of 6-BAP in combination with one of the auxins IAA, IBA, NAA or 2,4-D. The cytokinin: auxin ratio in the medium was 10:1 or 20:1. The best regeneration of Heuchera hybrid (up to 92% of regenerating leaf discs) was obtained on media containing 6-BAP at a concentration of 4.0 mg/l in combination with NAA at a concentration of 0.2 mg/l or 0.4 mg/l. In this case, the number of adventitious shoots per regenerating disc could reach 7‑9 and more. The maximum frequency of direct regeneration was observed on media containing IBA. Abundant callus formation was observed on media containing 2,4-D.

Neem Oil to Reduce Zeatin Use and Optimize the Rooting Phase in Olea europaea L. Micropropagation.

Micropropagation is an in vitro propagation technique, established in the nursery field sector for numerous species, which offers several advantages compared to traditional agamic propagation techniques. In the case of the olive tree, however, despite the advances made through research, it is still little used, due to the recalcitrance to in vitro proliferation and/or rooting of many olive cultivars and the high cost of zeatin, the only cytokinin that makes it possible to achieve a satisfactory proliferation rate in this species. In this context, numerous attempts have been made to identify alternative cytokinin compounds able to improve the proliferation rate of olive tree explants and thus reduce the unitary production cost. In particular, there is a growing interest in the use of natural substances (called in some cases "complex mixtures"), which, when added to the in vitro cultivation substrates, seem to be able to improve proliferation rates. In the present study, neem oil was added to the propagation substrates (partially/totally replacing zeatin) and in the rooting phase for the olive cultivar Moraiolo. In particular, in the proliferation phase, the effect of neem oil (0.1 mL L-1) in substrates containing different zeatin concentrations (0, 1, 2, and 4 mg L-1) was evaluated. For the rooting phase, agarized substrate and soil were used with shoots derived from a standard proliferation substrate (4 mg L-1 zeatin) and from the substrate that gave the best results in the proliferation phase (2 mg L-1 zeatin and 0.1 mL L-1 neem oil). In the proliferation phase, the addition of neem oil in the substrates with low zeatin concentration (1 and 2 mg L-1) induced an increase in the number of adventitious shoots and shoots length. On the contrary, the addition of neem oil in the rooting substrates did not positively influence the rooting phase, but positive results especially in terms of root number and length were observed in explants derived from a neem oil-enriched proliferation substrate compared to the control substrate. Therefore, the present study demonstrated for the first time the positive role of neem oil in the proliferation of olive in vitro with low zeatin concentrations.

In vitro propagation, shoot regeneration, callus induction, and suspension from lamina explants of Sorbus caloneura.

Sorbus caloneura has high ornamental and medicinal value but is endangered, and significant effort is required to preserve this natural resource. In this study, the stem and sterilized leaves of S. caloneura were used to explore the effects of different plant hormones basic medium type, initial callus quality, initial liquid volume, and ratio of old liquid culture medium to new on stem proliferation, regeneration and callus suspension culture. Naphthylacetic acid (NAA) and 6-benzyladenine (6-BA) significantly affected the proliferation of stem tissue. Murashige and Skoog (MS) basal medium supplemented with 1.75 mg/L 6-BA, 0.25 mg/L NAA, and 0.25 mg/L indole butyric acid (IBA) yielded a proliferation rate of 100%, with an average number of adventitious shoots per stem of 4.9. The best callus induction was observed with MS basal medium containing 0.5 mg/L 6-BA, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.2 mg/L thidiazuron (TDZ). The adventitious shoots were directly induced by MS basal medium containing 5.0 mg/L 6-BA, 0.5 mg/L NAA, and 1.5 mg/L kinetin (KT) and indirectly induced by medium containing 3.0 mg/L 6-BA, 0.3 mg/L NAA, and 0.1 mg/L TDZ. NAA significantly affected rooting rate, with ideal conditions found to be medium supplemented with 0.2 mg/L NAA and 1.5 mg/L IBA. MS basal medium with 4.5 g of calli and 120 mL of liquid culture medium without retention of the original culture medium yielded the best suspension effect for callus proliferation. Taken together, the results of this study lay a foundation for the breeding, preservation of germplasm resources, and genetic transformation of S. caloneura.

Regenerasi Tanaman Kitolod (Hippobroma longiflora (L.) G.Don) pada Kultur In Vitro

Kitolod (Hippobroma longiflora (L.) G. Don) is a wild plant. Its flower is widely used as a traditional medicine.When this plant is utilized more intensively, there may be a shortage of the plant due to the lack of seed sources. This study aimed to obtain the best techniques and culture conditions for in vitro propagation of kitolod to provide a large number of planting materials. The experiments were arranged using a completely randomized design with two treatment factors and 10 replications for all experiments except in shoot rooting. Leaves and petioles were used as explant sources. Various combinations of benzilamino purine (BAP) and naphtalene acetic acid (NAA) were applied. Leaf explants in Murashige and Skoog (MS) medium enriched with 1 mg/L BAP and 0.1 mg/L NAA combination produced the highest number of adventitious shoots per explant, but 2 mg/L BAP + 0.1 mg/L NAA was more effective for shoot initiation and multiplication. The latter medium was also able to produce the tallest shoots, and presented 75% of successful rate over the acclimatization period. The best rooting was provided by MS medium added with 0.5-1.0 mg/L NAA.

Micropropagation of Rare Scutellaria havanensis Jacq. and Preliminary Studies on Antioxidant Capacity and Anti-Cancer Potential.

We report the development of in vitro propagation protocols through an adventitious shoot induction pathway for a rare and medicinal Scutellaria havanensis. In vitro propagation studies using nodal explants showed MS medium supplemented with 10 µM 6-Benzylaminopurine induced the highest number of adventitious shoots in a time-dependent manner. A ten-day incubation was optimum for shoot bud induction as longer exposures resulted in hyperhydricity of the explants and shoots induced. We also report preliminary evidence of Agrobacterium tumefaciens EHA105-mediated gene transfer transiently expressing the green fluorescent protein in this species. Transformation studies exhibited amenability of various explant tissues, internode being the most receptive. As the plant has medicinal value, research was carried out to evaluate its potential antioxidant capacity and the efficacy of methanolic leaf extracts in curbing the viability of human colorectal cancer cell line HCT116. Comparative total polyphenol and flavonoid content measurement of fresh and air-dried leaf extract revealed that the fresh leaf extracts contain higher total polyphenol and flavonoid content. The HCT 116 cell viability was assessed by colorimetric assay using a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide, showed a steady growth inhibition after 24 h of incubation. Scanning electron microscopy of leaf surface revealed a high density of glandular and non-glandular trichomes. This research provides a basis for the conservation of this rare plant and future phytochemical screening and clinical research.

Strigolactone signaling inhibition increases adventitious shoot formation on internodal segments of ipecac.

SL inhibited adventitious shoot formation of ipecac, whereas the SL-related inhibitors promoted adventitious shoot formation. SL-related inhibitors might be useful as new plant growth regulators for plant propagation. In most plant species, phytohormones are required to induce adventitious shoots for propagating economically important crops and regenerating transgenic plants. In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), however, adventitious shoots can be formed without phytohormone treatment. Here we evaluated the effects of GR24 (a synthetic strigolactone, SL), SL biosynthetic inhibitors, and an SL antagonist on adventitious shoot formation during tissue culture of ipecac. We found that exogenously applied GR24 suppressed indole-3-acetic acid transport in internodal segments and decreased the number of adventitious shoots formed; in addition, the distribution of adventitious shoots changed from the apical to middle region of the internodal segments. In contrast, the SL-related inhibitors promoted adventitious shoot formation on both apical and middle regions of the segments. In particular, SL antagonist treatment increased endogenous cytokinin levels and induced multiple shoot development. These results indicate that SL inhibits adventitious shoot formation in ipecac. In ipecac, one of the shoots in each internodal segment becomes dominant and auxin derived from that shoot suppresses the other shoot growth. Here, this dominance was overcome by application of SL-related inhibitors. Therefore, SL-related inhibitors might be useful as new plant growth regulators to improve the efficiency of plant propagation in vitro.

Establishment of a micropropagation supporting technology for the Fraxinus mandshurica × Fraxinus sogdiana

The interspecific hybridization can take advantage of heterosis and combine the double parental traits most extensively, and is an effective strategy for improving species and stress tolerance breeding for cross-pollinated trees. Species of Fraxinus L. are important for landscaping, timber, and afforestation tree species. To solve problems of poor cold adaption associated with the introduction of Fraxinus sogdiana in Maoershan (Heilongjiang province, Northeast China), to improve pest resistance of Fraxinus mandshurica, and to take advantages of the characteristics of F. mandshurica and F. sogdiana, the interspecific hybridization of F. mandshurica × F. sogdiana was conducted. Tissue culture is the key technology for the promotion of excellent clones and is a powerful tool for germplasm conservation of endangered or valuable plant species. Basing on the embryo culture of hybridization seeds, a complete and efficient micropropagation technology of the Fraxinus mandshurica × Fraxinus sogdiana for its production was established by the uniform design, regression analysis, and verification tests for optimal conditions. In this micropropagation technology, induction rates of callus, adventitious shoot, and adventitious root were 83.5%, 65.9%, and 80.0%, respectively; the number of adventitious shoots was 12.5 produced per callus and the survival rate of the seedlings grew in the greenhouse could be 91.3% after acclimatization. These findings enriched the germplasm resources of Fraxinus species, supplied a micropropagation supporting technology for production of F1 hybrids with ideal traits and accelerated the genetic crossbreeding process of F. mandshurica.

In-vitro development of Nauclea diderrichii (de Willd. & Th. Dur) Merrin liquid-M Smedia supplemented with benzyl amino purine (BAP) and naphthalene acetic acid (NAA)

The growth of plantlets in Temporary Immersion Bioreactor system (TIBs) relies on initial successful liquid phase transition process. The response of N. diderrichii explants was assessed in liquid-M Smedia with a view to mass produce its seedlings using TIBs. Seven treatments consisting (A) 0.0/0.0, (B) 0.0/0.1, (C) 0.1/0.0, (D) 0.2/0.1, (E) 0.3/0.2, (F) 0.4/0.3 and (G) 0.5/0.4mg/lBAP/NAA combinations were studied. Each group consist of seven replicates and group A without Growth Regulators (GR) serves as control. The results at 4 Weeks after Inoculation (WAI) showed that effects of the growth regulators were significant on shoot length and number of adventitious shoots while number of roots and leaves were closely related. Treatment E produced highest number of adventitious shoots (3.6) which was higher than 0.9 shoots from treatment G and closely related to others. Maximum number of leaves (16.6) was produced by treatment F followed by E (15.7) while the least (12) was obtained in treatment A. The highest number of roots (4.9) was obtained from treatments B, followed by E (4.3) with the lowest being recorded in C (2.43). Liquid MS medium supplemented with 0.3/0.2mg/lBAP/NAA shows some promise for plantlets generation for the purpose of multiple shoot production of N. diderrichii in TIBs.

A novel regeneration protocol for LA Lilium

Lilium is a high value ornamental crop and its demand particularly LA hybrids is growing day by day. To utilize the high regeneration potential in Lilium, transverse thin cell layer sections were excised from in vitro bulb scales of LA Lilium hybrid cv. Brindisi and inoculated on MS medium with 3% sucrose, 0.8% agar and varied concentration of 6-BAP, NAA and 2,4-D. Results showed that Protocorm like Bodies (PLB’s) were directly being induced from the surface of t TCL explant. These protocorm like bodies are unipolar structure forming shoot and further developed root when transferred to rooting medium. When NAA was fortified with 6-BAP in MS medium, its regeneration percentage was recorded maximum in the combination 6-BAP (2 mg/l) and NAA (0.50) mg/l), whereas, maximum number of adventitious shoots per explant were recorded in 6-BAP (3 mg/l) and NAA (0.50) mg/l). Although, adventitious shoot buds were also produced on hormones free medium but at a very slow rate. The regeneration percentage was enhanced in the presence of NAA as compared to 2, 4-D when supplemented with 6-BAP. The regenerated bulblets were best rooted in IBA supplemented with ½ MS medium and hardened in the glass jar.

In vitro regeneration and flowering of Portulaca grandiflora Hook

Abstract P. grandiflora is a known ornamental plant with abundant flowering. The flowers exhibit varied coloration with distinct forms and simple folded petals and/or multiple. The objective of this work was to induce regeneration via organogenesis and in vitro flowering of P. grandiflora. Nodal segments of seedlings germinated in vitro were used as explant source for regeneration. Kinetin (KIN) and 6-Benzylaminopurine (BA) were used for the induction of organogenesis. The treatments supplemented with 1.0 and 1.5 mg L−1 BA induced the highest number of adventitious shoots with an average number of 7.0 (±1.55) e 5.4 (±0.83), respectively. The microcuttings obtained from regenerated shoots produced floral buds. The floral buds were located in the axillary and terminal regions of the microcuttings and developed in approximately 10 days of cultivation until the anthesis. The highest number of flower buds was observed in the presence of 0.75 mg L−1 of gibberellic acid. This study opens new perspectives for the establishment of biotechnological tools to be applied for this important ornamental species.

Reinvigoration of diploid strawberry (Fragaria vesca) during adventitious shoot regeneration

Diploid strawberry (Fragaria vesca ‘Baiguo’) is a model plant for studying functional genomics in Rosaceae. Adventitious shoot regeneration is essential for functional genomics by Agrobacterium tumefaciens-mediated transformation. An efficient shoot regeneration method using diploid strawberry leaf explants was conducted on 1/2MS + 1/2B5 medium that contained 2.0 mg L−1 TDZ over 14 days of dark culture; this induced the maximum percentage of shoot regeneration (96.44 ± 1.60%) and the highest number of shoots per explant (23.46 ± 2.14) after 11 weeks of culture. The explants considerably enlarged after 12 days; then, turned greenish brown after 30 days, yellowish brown after 36 days, and completely brown and necrotic after 48 days. Large numbers of adventitious shoots were produced from 48 to 66 days, and the shoots elongated from 66 to 78 days; this represents a critical period of reinvigoration, which included 30 days for leaf explant chlorosis, 36 days for adventitious shoot appearance, and 48 days for generation of numerous shoots. During the reinvigoration process, higher expressions of the hormone synthesis-related genes Ciszog1, CKX2, CKX3, CKX7, YUC2, YUC6, YUC10, YUC9, and GA2ox were detected from 30 to 48 days. Our results indicate that these genes may regulate reinvigoration of shoot regeneration.

PENGARUH PRA-STERILISASI TERHADAP KEBERHASILAN INISIASI EKSPLAN YANG BERASAL DARI PUCUK SENGON

Sengon known as forest product with high market demand . Tissue culture is one multiplication method to get seeds in large quantities . One factors that support the success of reproduction is the health of the plant material. The purpose of the study is to determine the influence of pre-sterilization treatments towards the successful initiation of sengon organogenesis. The research method was done by spraying the seedlings with Pyraclostrobin 50 g.kg -1 + Metiram 550 g.kg -1 of 2,500 ppm every week for 3 months. Each explant was grown on MS medium. The observation was carried out done every 1 week for 12 weeks, including the amount of contamination (bacteria, fungi), browning and the appearance of buds, roots and callus. Data were analyzed by t-test. The next stage is multiplication by using treatments of 24D 0.1 mM, 24D 0.5 mM, BAP 0.5 mM, BAP 2 mM, K 0.1 mM, K 0.5 mM, TDZ 0.1 mM, TDZ 0, 5 mM, and control. Completely randomized design was used and analyzed by using one way ANOVA, and tested with Duncan for the differences at 0,05 significant level. The results showed that pra-sterilization treatment was capable of supporting the successful initiation of sengon shoots characterized by low levels of contamination, increased number of callus and number of sengon shoots explants. Kinetin 0.01 mM in the multiplication media of sengon shoots was able to increase the number of adventitious shoots.

OBTAINING PLANT MATERIALS SIBERIAN IRIS (IRIS SIBIRICA L.) BY METHODS OF BIOTECHNOLOGY

Methods of biotechnology allow to obtain high-quality medicinal plant raw materials in a short time, in large quantities without destroying natural reserves. Biotechnological approaches such as aeroponic technologies have the potential for large-scale cultivation of iris plants and production of secondary metabolites. Microclonal reproduction makes it possible to obtain a healthy planting material in the required amount, regardless of the time of year. The combination of these two technological approaches will allow to develop biotechnology of year-round production of medicinal plant raw materials of Siberian iris.
 The study determined the content of 6-benzylaminopurine on the stage actually micropropagation for the formation of the greatest number of adventitious shoots of optimal length. The required content of BAP in the nutrient medium for I. sibirica was 2.5–5.0 µM. The introduction of cytokinins in the nutrient medium together with auxins, L-glutamine and adenine sulfate 100 mg/l, as well as the alternation of low and high concentrations of cytokinin enhanced the regenerative effect of BAP. With year-round cultivation of regenerative plants in aeroponic conditions, the amount of biomass of plant raw materials I. sibirica for this method was about 31.2 kg / m2 of crude weight in one year.
 It is established that intact plants and regenerative plants I. sibirica, obtained on the basis of the developed biotechnology, had identical group composition of biologically active substances. It is revealed that the sum of flavonoids in the leaves of hydroponic iris plants exceeded the content in the leaves of intact plants by 3 times, and the content of essential oil in regenerate plants and hydroponic leaves of the Sterch variety but higher by 26% compared with the leaves of intact plants.
 Aqueous and alcoholic extracts of I. sibirica showed antiviral activity against herpes virus. With low toxicity, both intact plants and regenerative plants had a relatively high selectivity index.

Comparative Study of In Vitro Root and Shoot Proliferation from the Node Explants of Asparagus racemosus Willd

Asparagus racemosus Willd. locally known as Kurilo or Shatavari is found throughout the tropical and subtropical regions of Nepal. It is a high value herb because of its medicinal and nutritional values which is leading this species decrease in its natural habitat. Hence, to conserve this species, an in vitro tissue culture method of its multiplication has been applied as an effective method of ex situ conservation. In the experiment, although most of the treatments induced highly insignificant number of adventitious shoots, low concentration of IBA (0.1-0.5 mg/l) in combination with relatively higher concentrations of Kinetin (1.0-2.0 mg/l) in MS medium were found to be significantly inducing up to 8.33±1.308 shoots/node. Among the treatments where hormones were used singly, IAA 0.5 and Kinetin 1.0 induced 4.83±1.08 and 4.66±1.43 shots/node respectively. Hence, a protocol for the effective multiplication of this species has been developed which can be used accordingly.

Induction of callus and adventitious shoots on epicotyl and hypocotyl segments of cumaru (Dipteryx odorata)

Somatic embryogenesis from callus induced in epicotyl and hypocotyl segments can be viable native species in order to better -benefit ratio costs, and rates of clonal multiplication. In this sense, two trials were established to induce callus and adventitious buds on hypocotyl and epicotyl segments of cumaru bean seedlings germinated in vitro in different concentrations and combinations of growth regulators. At first, we used the MS medium supplementwith ANA (0.0, 1.5 mg.L-1) and TDZ (0.0, 4.0 and 8.0 mg.L-1) distributed in factorial 2 x 3 x 2 (x auxin cytokinin x explant) with eight replications. In the second, it was used the WPM medium supplemented with BAP (2.0 mg L-1) and plus 2,4-D (2.0 and 4.0 mg L-1) in a factorial 2 x 2 (auxin x explant) with 15 repetitions each. They were evaluating callus formation and the average number of adventitious shoots during the period of 90 days. The results indicated that the highest average for callus formation was observed when the explants were subjected to concentrations of 8.0 mg L-1 TDZ combined with 1.5 mg L-1 ANA in MS medium. For the formation of buds, the WPM medium plus 2.0 mg L-1 2,4-D in the second experiment, induced higher number of shoots, being significant the use of auxin, and its interaction with the type of explant.

Adventitious shoot regeneration from in vitro leaves of Aronia mitschurinii and cotyledons of closely related Pyrinae taxa

The objective of this study was to develop an in vitro shoot regeneration procedure and to evaluate the frequency of adventitious shoot regeneration from: (1) in vitro leaves of a commercial cultivar of Aronia mitschurinii on various media treatments; (2) cotyledons of closely related Pyrinae taxa; and (3) 21 wild Aronia genotypes. Optimum regeneration of leaf explants occurred when they were wounded with two transverse cuts along the midrib and placed on Murashige and Skoog (MS) basal media containing 5 μM indole-3-butyric acid (IBA) and 10 μM thidiazuron (TDZ). TDZ was more effective than 6-benzylaminopurine (BAP) as a cytokinin, and IBA was more effective than the no auxin control, 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-naphthaleneacetic acid (NAA). Regeneration from cotyledons of seven Pyrinae taxa was evaluated using 10 μM BAP in combination with 0.1, 1 and 5 μM NAA. Adventitious shoot formation for A. melanocarpa and P. communis responded best to 1 μM NAA, whereas all other taxa formed a greater number of adventitious shoots on 5 μM NAA. A. mitschurinii cotyledon explants produced a significantly greater number of shoots compared with in vitro leaf explants. The number of shoots forming per cotyledon explant and the percent of explants forming shoots were both significantly different among the 21 Aronia genotypes. Significant differences were observed between the six Aronia taxonomic groups for the number of shoots forming per explant. Diploid and tetraploid Aronia genotypes produced a significantly greater number of shoots per explant than did triploid genotypes. Regenerated shoots were rooted in vitro and plants grew normally in the greenhouse. These results will be useful for future studies using leaf and cotyledon explants for genetic transformation, genome editing and mutation breeding with Aronia and related taxa.