In vitro regeneration through organogenesis of Meconopsis simplicifolia — An endangered ornamental species

Meconopsis simplicifolia (D.Don) Walp. could be propagated by induction of adventitious shoots from callus produced on hypocotyl, cotyledon and rosette leaf explants of 4-month-old seedlings. Callus was initiated on agar-solidified Murashige and Skoog medium supplemented with 1 M kinetin +10 M -naphthaleneacetic acid (NAA). Shoots formed when the callus was subcultured on medium supplemented with kinetin or benzyladenine (BA) in combination with NAA, indoleacetic acid, indolebutyric acid or gibberellic acid. Excised shoots were rooted on medium containing auxin with 10 M NAA producing the best rooting (55%).Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy-acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA -indolebutyric acid - NAA -naphthaleneacetic acid     

In vitro regeneration of plantlets in Fagraea fragrans Roxb. — a tropical tree

Different vegetative parts of Fagraea fragrans Roxb., a valuable timber tree of South East Asia, were used as explants in in vitro studies.Nodal segments showed the best growth response as numerous adventitious shoots were regenerated in MS medium supplemented with Benzyl adenine (BA, 8.8 um) and 2,4-dichlorophenoxyacetic acid (2,4-D, 0.5 um). Shoot buds also developed from the leaf and root segments that were subcultured. The detailed process of callus growth and differentiation leading to the formation of whole plantlets is described. Uniform plantlets obtained could be transplanted successfully in soil.     

Is the endangered Apium repens (Jacq.) Lag. rare because of a narrow regeneration niche?

The conservation of rare plant species is an important aspect of global biodiversity protection, but in many cases these species and the reasons why they are rare are poorly understood. The perennial umbellifer Apium repens is generally regarded to be a rare species all over its European range. We hypothesized that its rarity might be caused by a restricted regeneration niche, that is, highly specific requirements for sexual regeneration, low seed dispersal potential and low endurance capacity of seeds in the ground. We conducted several experimental investigations on its germination ecology, hydrochorous dispersal potential and soil seed bank properties. Apium repens showed high germination success under a variety of abiotic conditions. Either light or cold-wet stratification was necessary to stimulate germination. Seeds were able to float for more than 50 days when protected from precipitation, and soil seed bank sampling revealed that at least some seeds of A. repens were contained in soil depths of up to 10 cm. Overall, our findings do not support the hypothesis that the rarity of A. repens is caused by highly specific requirements for its sexual regeneration. Nonetheless, its germination ecology should be considered when designing conservation measures for this endangered species.     

In vitro propagation of Populus x wilsocarpa — a hybrid of ornamental value

Populus x wilsocarpa, a hybrid of important ornamental value, cannot be seed-propagated, nor grafted, since a compatible rootstock has not been identified. A micropropagation protocol consisting of a series of steps was therefore developed to facilitate the commercial production of this species. The technique involved the transfer of swelling buds to a growth initiation medium with the following composition: N6 macronutrients, MS micronutrients and vitamins supplemented with 0.5 mg l^-1 BAP. The best buds were from dormant twigs, stored at 0–2°C and then forced to burst prior to culture initiation. Shoot multiplication was on a basal WPM medium including 0.1 mg l^-1 BAP and 0.001 mg l^-1 NAA. Shoot elongation and rooting was also on a basal WPM medium supplemented with 1.0 mg l^-1 GA₃ followed by a transfer to a peat-perlite mix in the greenhouse.Abbreviations ABA abscisic acid - BAP benzylaminopurine - GA₃ gibberellic acid (GA₃) - MS Murashige and Skoog [17] - NAA naphthaleneacetic acid - N6 medium [Chu et al., 7] - WPM woody plant medium [16]     

In vitro micropropagation of a Cucurbita interspecific hybrid cultivar – a root stock plant

An efficient in vitro micropropagation protocol was developed for direct shoot growth of interspecific Cucurbita hybrid variety using shoot–tips of 5-day-old explants. The excised shoot–tips were cultured on Murashige and Skoog (MS) medium containing two plant growth regulators (6-benzyladenine and naphthaleneacetic acid) with various combinations and concentrations for the study of shoot induction. The best condition for shoot growth was with 3 mg l^–1 6-benzyladenine (BA) in MS medium. The shooting frequency was 84% and five shoots were obtained from each explant after 30 days of culture. Shoots (11.5 cm length) were rooted most effectively in 1 mg l^–1 indole-3 butyric acid (IBA)-supplemented MS medium. The highest root formation rate was 93% and all rooted shoots were transplanted into soil.     

In vitro anther culture of sweet orange (Citrus sinensis L. Osbeck) genotypes and of a C. clementina × C. sinensis ‘Hamlin’ hybrid

Citrus, and particularly sweet oranges, are very recalcitrant to anther culture. In this paper it was evaluated for the first time the response of 27 genotypes of Citrus sinensis and of one hybrid C. clementina × C. sinensis, to in vitro anther culture. Ten genotypes of sweet oranges showed embryogenic callus induction, mostly blood sweet oranges genotypes, such as Tarocco, Moro and Sanguinelli. In vitro microspore developmental switches from the gamethophytic to the sporophytic pathway were shown by DAPI staining in microspores of these responsive genotypes, after 10 months in culture. However, microsatellite marker analyses showed that these calli were heterozygous. The flow-cytometric analysis of these embryogenic calli showed the presence of two peaks, corresponding to haploid (n) and diploid (2n) genotypes. Differently, anther cultures of the hybrid C. clementina × C. sinensis produced tri-haploid (3n) embryogenic calli and the embryos obtained were homozygous when analyzed by molecular markers (sample sequence repeats), confirming the more responsive characteristic of clementine to microspore embryogenesis through anther culture.     

In vitro induction of secondary xylem-like tracheary elements in calli of hybrid poplar (Populus sieboldii × P. grandidentata)

The formation of tracheary elements was induced in calli derived from petioles of hybrid poplar (Populus sieboldii × P. grandidentata) after 10 days of culture on medium that lacked auxin but contained 1 μM brassinolide. Some differentiated cells formed broad regions of cell walls and bordered pits, which are typical features of tracheary elements of secondary xylem. Other differentiated cells resembled tracheary elements of primary xylem, with spiral or reticulate thickening of cell walls. The tracheary elements that developed in calli were formed within cell clusters. This induction system provides a new model for studies of the mechanism of differentiation of secondary xylem cells in vitro.     

In vitro regeneration of Alstroemeria cv. ‘Yellow King’ by direct organogenesis

The common techniques for the in vitro production of Alstroemeria plants are based on rhizomes as explants, which have low multiplication rates and a high risk of carrying viral diseases. To overcome these problems, we developed a protocol for the in vitro regeneration of Alstroemeria cv.‘Yellow King’, by testing for shoot induction several explant sources (leaf, stem apices, rhizomes and immature inflorescence apices), temperature and light/dark regimes, hormone and salt concentrations. For shoot multiplication and rooting, several hormone concentrations were tested. We found that only the young floral apices produced adventitious shoots by direct organogenesis. The highest shoot induction rate (10.4 shoots per explant) was obtained by incubation in the dark for 15 days at 8 °C followed by 15 days at 25 °C and a 16-h/8-h light/dark regime, on a Murashige and Skoog (1962) liquid medium at 50% of the salt concentration, supplemented with 2.5 mg l⁻¹ KIN, 1.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, using a piece filter paper to support the explant. The highest shoot multiplication rate (9 shoots per explant) was obtained on a liquid MS medium at full strength supplemented only with BA at 1.0 mg l⁻¹. In vitro rooting of shoots was induced also on a liquid MS medium, either with or without plant hormones.     

In vitro regeneration of plantlets from immature zygotic embryos of Picea chihuahuana Martínez, an endemic mexican endangered species

Summary Adventitious buds were induced from immature embryos of Picea chihuahuana, a species from the north of Mexico. The medium was Schenk and Hildebrandt supplemented with benzyladenine and kinetin, alone or in combination with naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid. Adventitious buds were obtained through a wide range of growth regulator concentrations, mainly from the cotyledonary region. Kinetin was more effective than benzyladenine in shoot induction. The optimum shoot induction medium contained 23.0 μM kinetin, on which 11.2 adventitious buds per embryo were obtained. Shoot development and elongation were achieved on basal SH medium at 50% concentration (SH 50) without growth regulators and improved with the reduction of sucrose concentration to 10 g l⁻¹. Differential response appeared to be associated with the provenance of the seeds, relative to the percentage of explants that responded as well as the number of adventitious buds produced per embryo. A low percentage of shoot rooting was obtained with a 24-h pulse on a medium with 16.1 or 26.85 μM of naphthaleneacetic acid. In the process of micropropagation of Picea chihuahuana, rooting is still the limiting factor.     

In vitro shoot growth of Brugmansia × candida Pers

The objective of this study was to improve the growth of in vitro shoot cultures of Brugmansia × candida ‘Creamsickle’. Several mineral nutrient experiments were conducted to determine the effect of NH₄⁺, NO₃⁻, K⁺, FeSO₄/EDTA, ZnSO₄, MnSO₄, and CuSO₄ on quality, leaf width and length, size and weight of shoot mass, and shoot number. The experiment to determine the levels of NH₄⁺, NO₃⁻, and K⁺, was conducted as a 2-component NH₄⁺: K⁺ mixture crossed by [NO₃⁻] and resulted in an experimental design free of ion confounding and capable of separating the effects of proportion and concentration. The results of the NH₄⁺-K⁺-NO₃⁻ experiment revealed a region in the design space where growth was significantly improved; the region generally had lower total nitrogen and lower NH₄⁺:K⁺ ratios than MS medium. The experiments to determine the appropriate levels of Fe, Zn, Mn, and Cu were conducted at six log levels ranging from 0 to 1 mM. Of the four metal salts tested, MnSO₄ had the least effect on in vitro shoot growth and its concentration was reduced from 0.1 mM (MS level) to 0.001 mM. CuSO₄ had large effects on in vitro shoot growth and was increased from 0.0001 mM to 0.001 mM. A 2-level factorial of NH₄⁺-K⁺-NO₃⁻, FeSO₄/EDTA, and ZnSO₄ was conducted and several formulations identified for their improvements of quality and growth. In addition to the changes to MnSO₄ and CuSO₄, these formulations were characterized by lower levels of NH₄⁺, K⁺, NO₃⁻ and Zn, and higher levels of FeSO₄/EDTA. Overall, several nutrient formulations were identified as superior to MS medium for growth of in vitro shoot cultures of B. ‘Creamsickle’.     

In vitro propagation of Syzygium travancoricum Gamble – an endangered tree species

Rapid multiplication in Syzygium travancoricum, an endangered tree species was achieved from nodal explants on Murashige and Skoog's (MS) medium supplemented with Woody Plant Medium (WPM) micronutrients and plant growth regulators. Multiple shoot induction from young 1–2-year-old seedlings was observed on basal medium supplemented with 17.7 M benzyladenine and 1.3 M -naphthalene acetic acid. A high number of multiple shoots (25 shoots/nodal explant) was observed by the third subculture on multiplication medium. The dark brown nodular callus obtained from in vitro grown shoots through axillary buds regenerated at a high frequency when transferred to 1/2 strength basal medium. The plants rooted on 1/2 strength basal medium supplemented with 1.1 M indole-3-acetic acid and were transferred to the field with 40% survival.     

In vivo observations of nucleolar extrusion in cultured callus cells of a non tumorousNicotiana glauca ×N. langsdorffii hybrid

Summary In vivo observations ofNicotiana glauca ×Nicotiana langsdorffii hybrid callus cells cultured on hanging-drop slides show a continuous pulsating movement of nucleoli containing vesicles from the nucleus into the cytoplasm and the occasional release of the nucleoli into the cytoplasmic stream. The phenomenon is discussed in relation to similar findings in other plant materials, particularly in critical phases of development.Contribution No. 151 from the Laboratorio di Mutagenesi e Differenziamento, Consiglio Nazionale delle Ricerche (C.N.R.), Pisa, Italy. Investigation supported by Consiglio Nazionale delle Ricerche (C.N.R.) under the programme Biology of Reproduction.     

In vitro plantlet regeneration of “dwarf” Indian olive (Elaeocarpus robustus Roxb.): a fruit plant of Bangladesh

A plantlet regeneration protocol was developed on pot-grown mature plants of Elaeocarpus robustus Roxb. cv. Dwarf from nodal and leaf explants. The best yield of adventitious shoots was achieved from the leaf-derived calli in a modified MS (MMS₁, half strength of major salts, full strength of minor salts, and vitamins) medium containing 4.0 μM BA + 4.0 μM Kn + 0.5 μM NAA + 15% coconut water (CW). The shoot multiplication rate was amplified about twofold per culture after the addition of 15% CW to the medium. The rate of shoot multiplication reached maximum at the 5th subculture, and it maintained this rate throughout the 3 subsequent subcultures. The best rooting in vitro was investigated by subculturing the microcuttings in an MMS₂ (half strength of both major salts and minor salts and full strength of vitamins) medium containing 1.0 μM IBA in the dark for one initial week at 30°C, followed by subculturing them in a plant-growth regulator (PGR)-free medium in the light. The plantlets raised in vitro were successfully established under ex vitro conditions.     

In vitro separation of a rose chimera

“Fairmount 1 thorny” (“FM1 thorny”) (a Rosa multiflora Thunb ex. J. Murr.) and a thornless sport of “FM1 thorny” (“Fairmount 1” (“FM1”)) were established in vitro to investigate chimeral segregation under various levels of BA and to obtain a pure thornless rose. While the chimeral thornless sport was expected to segregate in vitro and yield both thorny and thornless plantlets, “FM1 thorny” was to yield only thorny plants. “FM1” segregated in vitro into its constituent genotypes and yielded thorny and thornless plantlets, suggesting that “FM1” is chimeral. “FM1 thorny” produced only thorny plants in vitro. These results indicate that the “FM1 thorny” clone was not chimeral (pure thorny) and that the thornless regenerates of “FM1” did not develop via somaclonal variation. There was a significant linear relationship between increasing BA concentration and the percentage of thorny plants. Among a population of 690 tissue culture derived plants from all the BA experiments, 6 plants were classified as pure thornless plants 1 year later.     

In vitro plantlet regeneration of Capsicum chinense Jacq. cv. ‘Bhut jalakia’: hottest chili of northeastern India

Chili (Capsicum chinense) cv. ‘Bhut jalakia’ is used in India for extraction of oleoresin and capsaicin as it is characterized by a very high capsaicin content. The conventional method of propagation of ‘Bhut jalakia’ is through seeds, but this is beset by short viability and low germination rates. Developing a suitable regeneration protocol for ‘Bhut jalakia’ was the focus of this study; as to date, in vitro regeneration for this cultivar has not been investigated. Cotyledon and shoot tip explants were cultured on Murashige and Skoog (MS) media supplemented with different concentrations of cytokinins and auxins. In the case of cotyledon explants, MS medium supplemented with 6-benzylaminopurine (BAP) at 35 μM and kinetin (KIN) at 15 μM were found to be optimal (4.00?±?0.57) for induction of multiple shoots per explant, whereas BAP at 14.8 μM and KIN at 60 μM were best (5.00?±?0.57) for growth of shoot tip explants. Shoots developed from cotyledon explants produced the maximum (8.67?±?0.32) number of roots on MS medium supplemented with low concentration (2.6 μM) of 2-naphthaleneacetic acid (NAA). Supplementation of indole-3-butyric acid (IBA) at 5 μM was found optimal for root formation (16.67?±?2.60) for shoots derived from of shoot tip explants. One month after transfer of in vitro regenerated plantlets to various potting mixes, the highest survival rate (40%) was observed in a mixture of sand, soil, and cow dung in a ratio of 1:1:1. Thus, both shoot tip and cotyledon explants may be cultured on MS medium modified with BAP, IBA, NAA, and KIN to regenerate ‘Bhut jalakia’ chili plants within 90 d.     

Adventitious bud regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Eucalyptus urophylla × E. tereticornis interspecific hybrid

In Vitro Cellular & Developmental Biology - Plant - A high-efficiency regeneration and genetic transformation system is indispensable for generating desirable traits in important trees such as...     

Electrophoretic confirmation of the intergeneric hybrid ×Ruttyruspolia (Acanthaceae)

A plant collected in South Africa in the early 1960's has been considered an intergeneric hybrid with the parental taxa beingRuspolia hypocrateriformis (Vahl)Milne-Redhead var.australis Milne-Redhead andRuttya ovata Harv. The intermediate morphology of the plant provided the strongest evidence of its hybrid origin. The natural hybrid, named formally as ×Ruttyruspolia A. Meeuse & de Wet, is highly sterile. Crosses between the two presumed parental taxa produced two plants that are very similar to the putative natural hybrid. We had examined the presumed parental species and the natural and artificial hybrids using enzyme electrophoresis. The two parental species are highly differentiated at genes specifying soluble enzymes; they have a genetic identity of 0.51. They have no common alleles at two genes, and contain alternative alleles in very different frequencies at two loci.Ruttya andRuspolia exhibit both unique and common alleles at two additional genes. The natural and artificially produced plants of ×Ruttyruspolia are identical electrophoretically and contain alleles unique to each of the parental species at two genes. In addition, individuals of ×Ruttyruspolia combine alternative high frequency alleles from each parent at two loci. Allozymes provide strong confirming evidence for the hybrid origin of naturally occurring ×Ruttyruspolia because the products of specific alleles either unique to or highly characteristic of the two putative parental taxa are found combined in ×Ruttyruspolia.     

Morphological characterization of a brown lemur hybrid zone (Eulemur rufifrons × E. cinereiceps)

Hybridization has recently been identified as a pervasive force in the evolution of primates. In this study, we characterized a hybrid zone between two species of brown lemur (Eulemur rufifrons and E. cinereiceps) in the Andringitra region of southeastern Madagascar using morphological traits. We immobilized animals along a north-south transect (～80 km), scored them for their degree of hybridity using pelage traits and measured standard morphometric variables. Results from our study suggest that hybridization between E. rufifrons and E. cinereiceps is extensive, with the hybrid zone extending over 42.6 km and being composed mostly of later generation hybrids. We also identified significant variation between ancestral groups in our study: hybrid males exhibited longer tails than both parental species and sexual dimorphism in upper canine height favoring males was documented in E. rufifrons. These patterns could suggest that gene flow between parental and hybrid populations is relatively limited. Finally, significant differences between ancestral groups in relative body mass and skin-fold thickness were absent in our study, indicating that, as measured by these proxies, hybrids are equally as fit as parental forms. Based on these preliminary findings, the Andringitra hybrid zone could conform to the bounded superiority model of hybrid zone stability (i.e., it could be being maintained by selection favoring hybrids within transitional habitats). Accordingly, hybrids in Andringitra may be an unusual case among primates, representing a stable recombinant but distinct lineage. This conclusion has important implications for evolutionary processes within the brown lemur species complex.     

In vitro establishment and multiplication of Nymphaea hybrid ‘James Brydon’

Rhizome tips were the most suitable explants for in vitro plant regeneration and multiplication of Nymphaea hybrid James Brydon on Murashige and Skoog medium containing different concentrations and combinations of indole-3-acetic acid, 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid, 6-benzyladenine (BA), kinetin, 2-isopentenyladenine (2iP) and gibberellic acid (GA₃). A combination of 2iP, BA, and NAA strongly favored induction of shoot buds and shoot proliferation. Pretreatment of shoot cultures at 8°C for 30 days or with 14.4 or 28.9 M GA₃ for 15 days did not improve shoot multiplication. A 16-h photoperiod with photosynthetic photon flux of 30 mol m^-2 s^-1 was found to be the optimum light condition for shoot growth and multiplication. Multiple shoots produced well developed root systems within 4 weeks after transfer to a plant growth regulator-free medium containing activated charcoal.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid