Propagation of an elite high biomass-producing genotype of Atriplex canescens by axillary enhancement

A technique for rapid in vitro multiplication and rooting of Atriplex canescens microshoots and establishment of plantlets in the soil is described. The influence of growth regulators in the multiplication medium on shoot growth, rooting, and soil establishment of in vitro-derived plants is assessed. Rapid propagation was achieved by the culture of shoot tips or lateral buds on a medium supplemented with 1 mg per liter of benzyladenine. Shoots were rooted in vitro on a medium supplemented with 1 mg per liter indolebutyric acid prior to placement in the soil. Plants were established successfully in the greenhouse through the maintenance of a high humidity environment for the first 21 days and were subsequently transferred to the greenhouse and field.     

Micropropagation of <Emphasis Type="Italic">Hagenia abyssinica</Emphasis>: a multipurpose tree

An in vitro propagation protocol has been developed for Hagenia abyssinica using original material from both juvenile and mature trees. Juvenile explants were obtained from seedlings, as well as shoots and meristems from 5 to 7-month-old greenhouse grown plants. Shoots collected from stem bases of five genotypes were used to establish cultures from mature trees. Explants of seedling origin were used to optimize the multiplication medium and growth regulators concentration. The best result was obtained from shoots subcultured on either MS or WPM medium supplemented with 4.4 M BAP and 0.49 M IBA. The initiated shoots from all the different explants were multiplied on these media. Rooting of shoots was achieved using MS medium containing macronutrients at one-third strength supplemented with 4.9 M IBA. The shoots were kept in the dark for 4 days and transferred to medium of the same composition but containing 0.3% activated charcoal without growth regulators. Up to 100% rooting was achieved depending on genotype. Shoots multiplied on MS medium rooted better than those multiplied on WPM. Plantlets were transferred to pots containing a mixture of soil and perlite in a 2:1 ratio, respectively, and were maintained in the greenhouse. Increased irradiance reduced stem and leaf lengths and increased branch number of micropropagated plants.     

Shoot organogenesis and plant regeneration in <Emphasis Type="Italic">Pityopsis ruthii</Emphasis>

Pityopsis ruthii is an endangered herbaceous perennial species from the United States. In vitro multiplication of this species can be valuable for germplasm conservation. Flower receptacles of P. ruthii were cultured on Murashige and Skoog medium (MS) supplemented with 11.4 μM indole-3-acetic acid (IAA) in combination with 2.2, 4.4 or 8.8 μM 6-benzyladenine (BA). Shoots were visible within 14–28 days and three plants were successfully rooted on MS medium supplemented with 5.7 μM IAA. A two tailed t-test for paired-variates revealed that shoot regeneration on MS medium amended with 11.4 μM IAA and 2.2 μM BA was significantly higher (P < 0.05) than on other treatments. Leaf explants were also cultured on MS not supplemented with growth regulators or supplemented with 11.4 μM IAA in combination with 0, 2.2, 4.4 or 8.8 μM BA. Shoots were visible within 21–35 days and one plant was successfully rooted on MS medium supplemented with 5.4 μM NAA. Shoot regeneration on MS medium augmented with 11.4 μM IAA and 2.2 μM BA was significantly higher (P < 0.05) than the other treatments according to analysis of variance (ANOVA) with a rank transformation. Hyperhydricity and rooting of shoots was problematic for explants derived from flower receptacles and leaf tissue, but viable plants were regenerated using both explants sources indicating the potential role for micropropagation in the ex situ conservation of the species.     

In vitro propagation ofSalix tarraconensis Pau ex Font Quer,an endemic and threatened plant

Summary Salix tarraconensis Pau ex Font Quer, an endemic willow species from northeast Spain, was micropropagated with nodal segments. Shoot multiplication was obtained with different cytokinins, either on Murashige and Skoog medium or woody plant medium. Best results for shoot formation were obtained on Murashige and Skoog medium containing 4.9 μM of 6-γ-γ-dimethylallylaminopurine. Shoots showed strong apical dominance, and some cultures displayed apical necrosis. Benzyladenine gave the worst results; shoots displayed very slow growth, deformed leaves, and hyperhydrity. Good rooting of shoots was obtained with different auxins or without plant growth regulators on woody plant medium. The best results (90-100%) were obtained within 20 d. On rooting media with indole-3-butyric acid or indoleacetic acid, shoot elongation was good (35-40 mm length). Apical necrosis was observed in elongating shoots on rooting medium, but this disturbance favored axillary bud sprouting and formation of new shoots. Shoot length and quality of roots decreased gradually as the concentration of naphthaleneacetic acid increased. Plant survival was 90% 4 weeks after removal fromin vitro conditions.     

Regeneration of <Emphasis Type="Italic">Prunus salicina</Emphasis> Lindl (Japanese plum) from hypocotyls of mature seeds

Plant regeneration of Prunus salicina (Japanese plum) using mature seeds was studied and evaluated. Shoots were effectively induced from hypocotyl slices of mature seeds on media containing cytokinins. Among three plant growth regulators evaluated, thidiazuron (TDZ) was the most effective for shoot induction overall. Shoots were also induced using 6-benzylaminopurine (BA), but the effectiveness was reduced at low concentrations. Low regeneration was induced using kinetin. Three plum varieties were evaluated and the regeneration appeared to be genotype dependent. Induced shoots elongated, roots formed, and plantlets developed upon transfer of the shoots to the rooting medium. Primary shoots, when sub-cultured on fresh induction medium, produced multiple shoots, and such multiplication could continue for more cycles. The plantlets were transferred to soil, and the full plants were readily recovered in a greenhouse. The regeneration process was relatively fast as plants could be recovered in 4 to 5 mo. after the culture initiation.     

Micropropagation of <Emphasis Type="Italic">Pongamia pinnata</Emphasis> through enhanced axillary branching

A complete protocol is presented for the first time for the micropropagation of Pongamia pinnata, a biofuel tree, using cotyledonary nodes derived from axenic seedlings. Multiple shoots were induced in vitro from nodal segments through forced axillary branching. Murashige and Skoog (MS) medium supplemented with 7.5 μM benzylaminopurine (BAP) induced up to 6.8 shoots per node with an average shoot length of 0.67 cm in 12 d. Incorporation of 2.5 μM gibberellic acid (GA₃) in the medium during the first subculture after establishment and initiation of shoot buds significantly improved the shoot elongation. Single use of GA₃ during the first subculture eliminated the need for prolonged culturing on BAP medium. Further use of GA₃ in the medium was not useful. Shoot culture was established for at least two subcultures without loss of vigor by repeatedly subculturing the original cotyledonary node on shoot multiplication medium followed by shoot elongation medium after each harvest of the newly formed shoots. Thus, from a single cotyledonary node, about 16–18 shoots were obtained in 60 d. Shoots formed in vitro were rooted on full-strength MS medium supplemented with 1.0 μM indole butyric acid (IBA). Plantlets were successfully acclimated, established in soil, and transferred to the nursery.     

Effect of triacontanol on the micropropagation of <Emphasis Type="Italic">Costus speciosus</Emphasis> (Koen.) SM. Using rhizome thin sections

Summary The highest percentage of shoot regeneration of Costus speciosus was achieved using thin rhizome sections and triacontanol (TRIA). Factors affecting the rate of shoot multiplication and rooting with TRIA have been investigated. Initiation of shoot buds was observed when rhizome thin sections were cultured on B₅ basal medium supplemented with 5μgl⁻¹ TRIA. Shoots with two to three leaves produced roots when cultured on B₅ basad medium supplemented with 2 μgl⁻¹ TRIA. The well-rooted shoots were hardened and transferred to soil where they showed normal growth and a 100% survival rate was achieved. Results of this study showed that TRIA can be used as an effective growth regulator in the micropropagation and conservation of C. speciosus.     

Micropropagation of meadowfoam (Limnanthes spp.)

Summary A rapid micropropagation system was developed for meadowfoam (Limnanthes spp. Brown) using four genotypes of three species. Murashige and Skoog (MS) medium supplemented with N⁶ benzyladenine (BA) and indole-3-acetic acid (IAA) at 0, 0.1, 0.5, 1.0 and 2.0 mg/l was tested for multiplication, shoot elongation and rooting. Expiants were taken from pot-grown plants. The most useful level for shoot growth and multiplication of both floral induced and non-induced plants was 0.5 mg/l BA. IAA failed to affect shoot growth or multiplication. Expiants from non-induced plants multiplied at moderate to high rates on 0.5 mg/l BA, while those from induced plants multiplied slowly and tended to elongate and flower. Non-induced plants on 2 mg/l BA produced large numbers of tiny shoots; induced plants did not respond. Shoots of all genotypes rooted on MS medium without hormones and all plants grew normally after transplanting to soil. This system provides a new tool for the development of meadowfoam as a crop plant.Abbreviations (BA) N ⁶ -benzyladenine - (IAA) indole-3-acetic acid - (MS) Murashige and Skoog medium, 1962     

Direct shoot organogenesis from needles of three genotypes of <Emphasis Type="Italic">Sequoia sempervirens</Emphasis>

Using in vitro-grown needles of Sequoia sempervirens (D. Don.) Endl., direct shoot organogenesis was induced. The effects of three genotypes and two cytokinins, N⁶-benzyladenine (BA) and N-benzyl-9 (2-tetrahydropyranl) adenine (BPA), in combination with 2,4-D were investigated. Among tested cytokinins, BPA produced the highest frequency of shoot organogenesis from all three genotypes tested. Adventitious shoots were induced directly from explants without intervening callus within 5weeks following incubation. Shoots were elongated on a 1/2 Wolter and Skoog (WS) medium supplemented with activated charcoal but without growth regulators. Later, elongated shoots were transferred to a 1/4 WS medium, but without activated charcoal and free of plant growth regulators to promote continued shoot growth. These shoots rooted spontaneously.     

A protocol for Ulmus minor Mill. micropropagation and acclimatization

Here we report the establishment of a simple protocol for the micropropagation and acclimatization of U. minor. Branches with dormant buds were collected from mature elms and sprouted in a greenhouse. Tip and node segments were used as starting material for in vitro proliferation in a medium (designated here as DKW1) already used for the micropropagation of a clone of the English Elm (U. procera SR4). In the first assay, in which explants from nine different trees were used, 88.5% of the tip segments produced new axillary shoots thus proving to be the best explant type. Afterwards, material from four different trees (F4, F7, F13, F14), that had the highest sprouting rate in the greenhouse, was used to test for genotype influence. F14 proved to be the best genotype in culture and it was used for all the subsequent experiments. Shoots from F14 were used to assay in vitro rooting using five DKW based media. Rooting percentages were high for all media and varied between 80% and 100%. For acclimatization two approaches were assayed: the use of previously rooted in vitro plants and the direct acclimatization of shoots from cultures in DKW1. After 6 weeks, 86.4% of the in vitro rooted plants were successfully acclimatized and a slightly higher value, 88.6%, was attained by direct acclimatization of shoots with thick stems and hard leaves. These results proved that there is no need for a previous in vitro rooting step and that direct acclimatization can effectively reduce time and costs. Thus U. minor micropropagation and acclimatization can be divided into only two steps: proliferation of shoots in DKW1 and direct acclimatization of these shoots in a sterile soil mixture.     

Elongation, rooting and acclimatization of micropropagated shoots from mature material of hybrid larch

Factors were defined for elongation, rooting and acclimatization of micropropagated shoots ofLarix x eurolepis Henry initiated from short shoot buds of plagiotropic stecklings serially propagated for 9 years from an 8-year-old tree. Initiation and multiplication were on Schenk and Hildebrandt (SH) medium supplemented with 5 M 6-benzyladenine (BA) and 1 M indole-butyric acid (IBA). Stem elongation was obtained in 36% of the shoots on SH medium containing 0.5 M BA and 63% of the remaining non-elongated shoots initiated stem elongation after transfer on SH medium devoid of growth regulators. Rooting involved 2 steps: root induction on Campbell and Durzan mineral salts and Murashige and Skoog organic elements, both half-strength (CD-MS/2), supplemented with 1 M of both naphthaleneacetic acid (NAA) and IBA, and root elongation following transfer to CD-MS/2 medium devoid of growth regulators. Repeating this 2-step sequence yielded up to 67% rooted shoots. Acclimatization of plantlets ranged from 83% to 100%. Over 300 plants were transferred to the greenhouse; some showed plagiotropic growth.     

Micropropagation of <Emphasis Type="BoldItalic">Embelia ribes</Emphasis> Burm f. through proliferation of adult plant axillary shoots

Micropropagation of Embelia ribes was achieved through proliferation of axillary shoots obtained from mature plants. Nodal shoot segments, collected March–May, exhibited high-frequency (75%) shoot initiation when cultured on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ) at 1.13 μM and indole-3-butyric acid (IBA) at 0.49 μM. Subculture of sprouted shoots from the original explants on medium containing TDZ (1.13 and 0.45 μM) during the first and second subcultures was found essential for further shoot proliferation, while inhibition of shoot elongation by TDZ could be overcome by transferring shoot cultures onto MS medium containing 6-benzylaminopurine (BAP; 11.10 μM) for the third subculture. Treating the explants with an antioxidant mixture of 568 μM ascorbic acid, 119 μM citric acid, and 307 μM glutathione prior to inoculation, coupled with subculture at 2-wk intervals onto fresh medium, both helped to reduce browning of the explants and facilitated production of five to six shoots/explant. MS medium supplemented with BAP (4.44 μM) and IBA (0.49 μM) induced shoot multiplication, producing five to six shoots/explant with a shoot length of 3 to 4 cm over a 4-wk culture period. Shoots of 3 to 4 cm in length exhibited 100% rooting within 4 wk after transfer to media containing half the nutrient salt concentration of MS medium with 3.69 μM IBA. Ex vitro rooting in the greenhouse from the in vitro shoots treated with 4.93 μM IBA for 30 min exhibited 95% rooting in soilrite™ medium in a 4-wk period. About 85% of micropropagated plants were established successfully in root trainers. Three-month-old, hardened plants could further be successfully established in the field. In 1 yr, by using the above protocol, 3,200 plants could be produced from a single shoot and 2,700 could be established in the field.     

红厚壳茎段丛生芽诱导与植株再生

红厚壳(Calophyllum inophyllum)为藤黄科红厚壳属多年生木本植物,有很高的药用价值。该研究以红厚壳带节茎段为外植体,探讨生长调节剂对腋芽萌发及丛生芽诱导、伸长和试管苗生根的影响。研究结果表明,外植体腋芽萌发和丛生芽诱导效果最好的培养基是MS+NAA1.0+TDZ0.5,在此条件下培养21天后,转入添加0.5 g·L–1活性炭且无生长调节剂的MS培养基,可有效促进不定芽的伸长。将带不定芽的外植体先在附加1.0 mg·L–1NAA的1/2MS培养基上进行生根诱导4周,之后转入附加1.0 g·L–1活性炭的无激素培养基进行根的伸长培养,这样的两步生根法能有效促进红厚壳生根。     

Involvement of polyamines in the adventitious rooting of micropropagated shoots of the apple rootstock MM106

Apple rootstock MM106 shoots, raised in vitro, rooted at 96.7% after culture on a medium supplemented with an auxin for 5 d in darkness followed by culture on a second medium without growth regulators for 25 d in light. In control conditions (in absence of auxin in the first medium), these shoots did not root. Putrescine (PUT), spermidine (SPD), cyclohexylamine (CHA), and aminoguanidine (AG) enhanced rooting when applied during the first d of culture in the absence of IBA; on the contrary, α-difluoromethylornithine (DFMO) added to the first medium with IBA inhibited rooting. The endogenous levels of indole 3-acetic acid (IAA) and indole 3-acetylaspartic acid (IAAsp) increased up to a maximum concentration at days 2 and 3, respectively, in initial rooting conditions. PUT, when added with IBA, did not affect the typical IAA and IAAsp increase; when applied alone, it provoked an increase of their levels. Similar results were recorded with CHA. SPD, AG, and DFMO did not induce an increase of IAA and IAAsp in nonrooting conditions. The levels of endogenous PUT increased to a maximum at day 2 in rooting conditions; it was slightly affected by exogenous PUT and CHA application but reduced by SPD, AG, and DFMO. In rooting conditions, if the first medium was supplemented with SPD or AG, a small increase in peroxidase activity was observed, similar to that obtained with PUT treatment. The present work indicates an involvement of polyamines in the control of rooting and an interaction with auxins during the physiological phase of rooting. The consequence of this relationship was a different rooting expression, according especially to the content of these regulators in the culture medium.     

Morphophysiological in vitro performance of Brazilian ginseng (Pfaffia glomerata (Spreng.) Pedersen) based on culture medium formulations

Pfaffia glomerata has potential pharmacological and medicinal properties due to the production of a secondary metabolite known as the phytoecdysteroid 20-hydroxyecdysone (20E). There have been increasing efforts for massive in vitro propagation of Pfaffia plants due to high extractivism and overharvesting of this species. Research on the species has shown that photoautotrophic cultivation can improve the production of 20E. In addition, other abiotic factors such as the formulations of culture media can influence the morphophysiological behavior of the plants in vitro. Therefore, the objective of this study was to analyze the morphological and physiological performances of P. glomerata plants in different formulations of culture media, under photoautotrophic and photomixotrophic propagation conditions. Six medium formulations, the Driver and Kuniyuki medium (DKW), Correia et al. medium (JADS), Murashige and Skoog medium (MS), Quoirin and Lepoivre medium (QL), Rugini medium (OM), and Woody Plant medium (WPM), all supplemented with DKW vitamins, 100 mg L⁻¹ myo-inositol, 6.5 g L⁻¹ agar, and with or without 3% (w/v) sucrose, were evaluated. Cultures were maintained at 25 ± 2°C, with a 16 h-photoperiod under 60 μmol m⁻² s⁻¹ of irradiance under a fluorescent lamp for 50 d. Results showed that the presence or absence of sucrose, and the different nutritional formulations influenced growth, photosynthetic pigment content, endogenous levels of sugars, leaf morphology, levels of 20E, and transport of water and minerals in P. glomerata. Notably, OM, DKW, QL, and WPM media promoted higher production of 20E under photomixotrophic growth conditions.

     

Enhanced Efficiency of In Vitro Rootstock Micro-propagation Using Silica-Based Nanoparticles and Plant Growth Regulators in Myrobalan 29C (Prunus cerasifera L.)

Different experiments were conducted to establish and optimize an efficient in vitro micropropagation protocol for Myrobalan 29C rootstocks. Disinfection of initial explants with AgNPs (2.5%) reduced the needed amount of NaClO (5.0%) by half. The highest rates of induced active buds were obtained in the DKW (90.63%), MS (86.67%), modified MS (82.22%), and WPM (78.15%) culture media supplemented with BAP (2.22 μmol L^?1)?+?GA₃ (2.88 μmol L^?1)?+?IBA (0.05 μmol L^?1)?+?Fe-EDDHA (228.72 μmol L^?1). The highest quality of the proliferated shoots (5.0) was also achieved using DKW medium. Inclusion of GA₃ (5.76 μmol L^?1), Fe-EDDHA (114.36–228.72 μmol L^?1), or BAP (2.22 μmol L^?1) were also able to enhance the rate of shoot multiplication. Compared to the agar-solidified culture system, the established shoots proliferated more efficiently when immersed by bioreactor in the liquid DKW culture medium on a regular basis. Exogenous application of silica-based nanoparticles (NPs) including the chemically synthesized silica NPs (TSiO₂ NPs, 1.0 ppm), rice husk derived biogenic silica NPs (RSiO₂ NPs, 10.0 ppm), or amine modified silica NPs (ASiO₂ NPs, 10.0 ppm) to the multiplication medium increased the number of regenerated lateral shoots by 520%, 360%, and 349%, respectively. Proliferated shoots with well-developed root system were obtained from the rooting medium supplemented with 19.68 μmol L^?1 IBA. Our results indicated that the rootstocks of Myrobalan 29C could be efficiently propagated under in vitro condition providing proper culture medium and optimal concentrations of additives and plant growth regulators were adopted.

     

In vitro propagation ofAtractylodes lancea

Shoot cultures ofAtractylodes lancea DC. (Compositae) have been established by inoculating the flower bud on Linsmaier-Skoog's medium supplemented with 10⁻⁵ M naphthaleneacetic acid and 10⁻⁵ M 6-benzyl-aminopurine. Shoots were multiplied on a medium containing 10⁻⁶ M 6-benzylaminopurine. Propagated shoots rooted on a medium devoid of any plant growth regulators were transferred to potting soil and finally to the field.     

<Emphasis Type="Italic">In vitro</Emphasis> rooting of two <Emphasis Type="Italic">Eucalyptus urophylla X Eucalyptus grandis</Emphasis> mature clones

Abstract   The rooting capacity of microshoots derived from two mature Eucalyptus urophylla X Eucalyptus grandis half-sib clones kept for 3 y under intensive micropropagation was assessed in different in vitro conditions. A first set of experiments established that clone 147 microshoots rooted earlier and in greater proportions, while producing more adventitious roots overall than their homologs from clone 149. Modifying the composition of the basal 1/2-MS-derived rooting medium by 1/4-MS or Knop macronutrients, or reducing sucrose concentration to 10 g l⁻¹ did not enhance the rooting rates. However, together with the growth regulators added, they had a significant effect on the number of adventitious roots formed. With rooting rates reaching 81%, the higher rootability of clone 147 over clone 149 was further confirmed by the second set of experiments with significant effects of the various auxins tested and strong clone × auxin interactions on the proportions of rooted microshoots and on the number of adventitious roots. The best rooting scores were given by 5 μM indole-3-butyric acid (IBA) and 12.5 μM 1-naphthaleneacetic acid (NAA), whereas the microshoots exposed to 5 or 12.5 μM indole-3-acetic acid (IAA) were less responsive. Lower light intensities did not improve significantly root capacities, although differences might exist according to the genotype. Overall, root and shoot elongation was stimulated by light. At the end of the experiment, the rooted microshoots were markedly taller than the non-rooted ones, with significant influences of auxins and light intensity, and to a lesser extent, of the genotypes.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.     

Factors affecting <Emphasis Type="Italic">in vitro</Emphasis> propagation and field establishment of <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>

The effect of plant growth regulators (PGRs), gelling agents, sucrose and heat shock on shoot multiplication, shoot growth, rooting and subsequent survival of Chlorophytum borivilianum Sant. et Fernand was evaluated. Benzyladenine (BA) was found to be better cytokinin over kinetin (KIN) for shoot multiplication. Sucrose concentrations from 116–290 mM in the basal medium (BM) promoted shoot multiplication. Heat shock (50 °C, 1 h) also promoted shoot multiplication at these sucrose concentrations on both BM medium and BM supplemented with 5.0 μM BA. Beneficial effect of sucrose was also observed on rooting of shoots on BM as well as BM supplemented with 5.0 μM indole-3-butyric acid (IBA). Phytagel as a gelling agent was found to be more effective for shoot proliferation and growth compared to agar. Amongst various soil mixtures tested, higher survival of plants was observed in soil containing Vermicompost. It was interesting to note that a maximum plant survival (> 95 %) was observed when plants were directly transferred to net-house (irradiance reduced to 50 % with green net, without humidity and temperature control) than poly-house (with humidity and temperature control). Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis of regenerated plants showed genetic similarity to mother plant.