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.     

Improvement of in vitro propagation of apricot cultivar ‘Bebecou’

Factors affecting successful establishment in vitro, rapid proliferation and rooting of apricot cultivar ‘Bebecou’ were studied. Ethanol and NaOCl were applied in several combinations for disinfection; chilling, plant growth regulators BA, IAA and GA₃, antibiotics, different culture vessels and systems of subculture were evaluated for the optimization of shoot proliferation and the auxins NAA and IBA were assessed for root induction. The highest number of new microshoots/explant (18.7) was obtained in a culture medium supplemented with 2.2 μM BA+0.57 μM IAA after 300 h of chilling. The effect of GA₃ (11.4 μM) on shoot proliferation was positive in combination with 4.4 or 8.9 μM BA. Shoot length and productivity were highest at 2.2 μM BA+11.4 μM GA₃+0.57 μM IAA and at 2.2 μM BA+0.57 μM IAA, respectively and decreased as cytokinin concentration increased. The antibiotic ‘Na-cefotaxime’ had a minimal impact on shoot growth when used at the lowest concentration (250 mg l⁻¹). Subculture every 2 weeks in a medium supplemented with 2.2 μM BA and 0.57 μM IAA was more efficient for shoot induction than alternation of 20 days culture in a propagation medium supplemented with 2.2 μM BA and 10 days culture in an elongation medium supplemented with 1.1 μM BA and 5.71 μM IAA. The highest number of roots/shoot (8.1) was recorded at 19.6 μM IBA.     

In vitro propagation of two Iranian commercial pomegranates (Punica granatum L.) cvs. ‘Malas Saveh’ and ‘Yusef Khani’

An efficient in vitro propagation is described for Punica granatum L. using shoot tip and nodal explants. The influence of two basal medium, WPM and MS, and different plant growth regulators was investigated on micropropagation of the Iranian pomegranate cultivars, ‘Malas Saveh’ and ‘Yousef Khani’. For proliferation stage, media supplemented with different concentrations (2.3, 4.7, 9.2 and 18.4 μM) of kinetin along with 0.54 μM NAA was used. WPM proved to be more efficient medium compared to MS. The best concentrations of kinetin were 4.7 μM for ‘Malas Saveh’ and 9.2 μM for ‘Yousef Khani’, resulting in the highest number of shoots per explants, shoot length and leaf number. For both cultivars, half-strength WPM medium supplemented with 5.4 μM NAA was most effective for rooting of shoots. Rooted plantlets were successfully acclimatized and transferred into soil. The micropropagated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the mother plants.     

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     

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 propagation for conservation of the rare date palm (Phoenix dactylifera L.) ‘Amri’ using immature inflorescence

In Vitro Cellular & Developmental Biology - Plant - Immature female inflorescence plays a significant role in date palm micropropagation because inflorescences are available with no practical...     

Stable propagation of ‘selfish’ genetic elements

Extrachromosomal or chromosomally integrated genetic elements are common among prokaryotic and eukaryotic cells. These elements exhibit a variety of ‘selfish’ strategies to ensure their replication and propagation during the growth of their host cells. To establish long-term persistence, they have to moderate the degree of selfishness so as not to imperil the fitness of their hosts. Earlier genetic and biochemical studies together with more recent cell biological investigations have revealed details of the partitioning mechanisms employed by low copy bacterial plasmids. At least some bacterial chromosomes also appear to rely on similar mechanisms for their own segregation. The 2 μm plasmid ofSaccharomyces cerevisiae and related yeast plasmids provide models for optimized eukaryotic selfish DNA elements. Selfish DNA elements exploit the genetic endowments of their hosts without imposing an undue metabolic burden on them. The partitioning systems of these plasmids appear to make use of a molecular trick by which the plasmids feed into the segregation pathway established for the host chromosomes.     

‘In vitro’ and ‘in situ’ decomposition of nuisance macroalgae Cladophora glomerata and Pilayella littoralis

The decomposition of two macroalgal species Cladophora glomerata (CHLOROPHYTA) and Pilayella littoralis (PHAEOPHYTA) was studied in the laboratory and field conditions. These species are known to cause the extensive macroalgal blooms in the whole coastal range of the Baltic Sea. The objective of the experiments was to determine decomposition rates of the macroalgae, follow the changes in tissue nutrient content and validate the role of benthic invertebrates in this process. In the laboratory conditions, the differences in the decomposition rates of the algae were mainly due to the oxygen conditions. The weight loss of C. glomerata was slightly higher in anaerobic conditions than in aerobic conditions. If 99% of initial dry weight of P. littoralis was lost in aerobic conditions then only 20% was lost in anaerobic conditions. In general, the loss of phosphorus and nitrogen in algal tissues followed the weight loss. As an exception, the amount of nitrogen changed very little during the decomposition of C. glomerata. In field conditions, the photosynthetic activity exceeded the decomposition rate of C. glomerata at lower temperatures in spring. The decomposition of P. littoralis was estimated at 49% of its initial dry weight. The addition of benthic invertebrates had no effect on the decomposition process. In summer, the decomposition rates were estimated at 65% for C. glomerata and 68% for P. littoralis being in the same order of magnitude as observed in laboratory conditions. If the decomposition of C. glomerata was faster at the end of the experiment, the most significant losses of weight of P. littoralis took place during the first 2 weeks of deployment. Idotea baltica significantly contributed to the loss of C. glomerata. The decomposition rate of P. littoralis was reduced by the presence of Mytilus edulis and increased by Gammarus oceanicus.     

In vitro propagation of Asparagus maritimus – A rare Mediterranean salt-resistant species

Asparagus maritimus L. Miller is a rare species growing of the Mediterranean region and is morphologically similar to A. officinalis. In order to establish an efficient in vitro propagation protocol, explants were excised from spear segments and cultured on Murashige and Skoog (1962) medium containing 3% sucrose and various concentrations of growth regulators. The best shoot initiation (3–4 per explant) was achieved on a medium containing 0.88 M N⁶-benzyladenine (BA), 0.93 M kinetin, 1.07 M -naphthaleneacetic acid (NAA) and 3.90 M ancymidol. Shoot initiation could also be achieved without ancymidol but the shoots were thinner and longer. A very high shoot multiplication rate was achieved on media supplemented with 3% sucrose, 1.07 M NAA, 0.93 M kinetin, 0.44 M BA and various concentrations of ancymidol. The lowest concentration of ancymidol (0.39 M) significantly promoted the highest shoot multiplication rate (11.9 shoots/crown). For root formation, media were supplemented with 6% sucrose, 1.07 M NAA and various concentrations of ancymidol. Rooting frequency increased with higher ancymidol concentration up to 5.07 M (82.0% rooting). The number of ex vitro shoots formed was strongly correlated (r=0.66) with the length of roots formed in vitro, which was the highest at a 1.95 M ancymidol.     

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 vitro propagation of Gentiana kurroo — an indigenous threatened plant of medicinal importance

Shoot multiplication of Gentiana kurroo Royle, a threatened medicinal plant species, was achieved in vitro using shoot tips and nodal segments as explants. Fifteen-fold shoot multiplication occurred every 6 weeks on Murashige and Skoog's medium (MS) containing 8.9 M benzyladenine and 1.1 M 1-naphthaleneacetic acid. Rooting was accomplished successfully in excised shoots grown on MS basal medium containing 6% sucrose.Abbreviations BA 6-benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - MS Murashige and Skoog's medium - NAA 1-naphthaleneacetic acid     

In vitro induction of tetraploids in cassava variety ‘Xinxuan 048’ using colchicine

Polyploids have great breeding value as they could have higher vegetative yields, higher qualities and greater tolerances against biotic and abiotic stresses. This research is focusing on in vitro colchicine-induced tetraploids in cassava. The survival rate of explants decreased with the increase of colchicine concentrations. Based on the survival rate, the treatment with 0.05 g l^?1 colchicine for 2 days was the best protocol for tetraploid induction in the cassava variety ‘Xinxuan 048’. The determination of ploidy levels showed that 28 autotetraploidy and 12 mixoploidy plantlets were obtained from 44 plantlets. Thus, 90.9% of the plants were variants. Significant differences in morphology and anatomy were found between the diploid and tetraploid plants. Tetraploid plants showed better photosynthetic capacities than the original diploid plants. The tetraploid cassava regenerated herein will enrich the germplasm spectrum and open a new arena for breeding novel triploids with elite cultivars by interploid crossing in the future.     

In vitro shoot regeneration from leaf discs of Betula pendula ‘Dalecarlica’ EM 85

The effect of zeatin, NAA (-naphthaleneacetic acid), putrescine and cefotaxime on the frequency of shoot regeneration from Betula pendula Dalecarlica EM 85 leaf discs has been examined. About 80% of leaf discs were induced to form adventitious shoots when the culture medium contained 45.6 moll^-1 zeatin and 0.1 mmoll^-1 cefotaxime. The addition of NAA to zeatin-containing media prevented shoot regeneration but stimulated root development directly from leaf tissues. Putrescine (0.1 mmoll^-1) and cefotaxime (0.1 mmoll^-1) could both significantly increase the percentage of leaf discs regenerating on optimal zeatin-containing media, and increase the number of shoots per regenerating disc.Abbreviations NAA -naphthaleneacetic acid - BAP 6-benzyladenine     

In vitro propagation of Dendrobium aphyllum (Orchidaceae)—seed germination to flowering

This communication describes asymbiotic seed germination, protocorm development, micropropagation and flowering in in vitro and hardened seedlings of Dendrobium aphyllum (Roxb.) C.E.C. Fischer. Effects of four culture media viz., Murashige and Skoog (MS); Phytamax (Sigma Chemical Co. USA; PM); Mitra et al. (M) and Knudson ‘C’ (KC), 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D), peptone and activated charcoal were studied on seed germination and protocorm development. Maximum germination (97 %) was recorded in PM basal medium. Peptone (2.0 gl^?1) remarkably enhanced germination percentage (100 %), vigorous growth, high survival and subsequent development of protocorms, while in activated charcoal the response was not encouraging. BAP improved germination percentage, however, 2,4-D showed noticeably low seed germination. The morphogenetic response of protocorms and nodal segments of in vitro raised seedlings varied depending on type of explants and concentrations and combinations of plant growth regulators used. Stout root system was induced in 1/2PM + 0.5 mgl^?1 IAA. Approximately 10 % of the in vitro raised plants (4–5 cm) with 3–4 leaves flowered in vitro irrespective of flowering season. The well-rooted plants showed 80 % survival under green house conditions and flowering was noticed after 5–6 months in 10 % of hardened plants.     

In vitro organogenic competence of different organs and tissues of lily of the valley ‘Grandiflora of Nantes’

The in vitro organogenic competence of rhizome and flower stalk discs, flower buds and meristems of lily of the valley (Convallaria majalis L.) Grandiflora of Nantes was studied. Except for the meristems, the other organs were all capable of organogenesis. Rhizome and flower stalk discs formed roots, while flower buds formed flower bud-like structures, and it appeared very difficult to modify the type of organogenesis. After six subcultures of the flower bud-like structures, a series of developmental stages were distinguished, i.e. regeneration of flower bud-like structures first, then leaf-like organs, and finally vegetative buds. This work pointed out the organogenic competence of the rhizome and flower stalk fragments for the first time.Abbreviations BA 6-benzyladenine - 2iP N-isopentenyladenine - NAA 1-naphthaleneacetic acid     

In vitro propagation of guayule (Parthenium argentatum) — a rubber yielding shrub

Nodal explants (0.5 to 0.8 cm long) isolated from 2-year old shrubs of guayule, Parthenium argentatum Gray, when cultured on MS medium supplemented with different concentrations of KN, BAP, 2,4-D, 2,4-D + BAP, NAA and NAA + BAP produced callus tissues and shoots simultaneously with varying frequencies. Shoots were regenerated with a high frequency (80–88%) from callus on MS medium containing NAA + BAP with or without glutamine. Addition of glutamine to these media improved considerably the number of shoots formed from a known amount of callus. Shoots could be regenerated from 200 day old callus cultures with a very high frequency but the organogenetic capacity declined thereafter. Increase in the concentration of sucrose (upto 4%) significantly enhanced the shoot forming ability of callus, but higher concentrations (6%) suppressed it. Rooting was induced only in dark when IAA, IBA and NAA were used, but 2,4-D could induce them both in light and dark. The system is suitable for the mass propagation of this important rubber yielding plant.Abbreviations MS Murashige and Skoog (1962) - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - KN Kinetin - BAP 6-Benzylaminopurine     

In vitro propagation of the relict laperinne’s olive (Olea europaea L. subsp. Laperrinei)

Olea europaea L. subsp. laperrinei (Oleaceae) is an endemic taxon of the mountainous regions of central Sahara, consisting of currently fragmented and small relict populations. The tree can propagate vegetatively or by seed, but no recent natural regeneration was observed in the Algerian massive populations, some of which are considered threatened with extinction. Sterile triploid individuals were also identified in some populations showing increasing vigour. As a result of its long persistence and despite its rarity, the Laperrine’s olive is an iconic component of Saharan mountain ecosystems. The aim of this study is to develop an efficient micropropagation protocol for both diploid and triploid Laperrine’s olive to safeguard and preserve this genetic resource. Best shoot propagation was obtained on a modified Murashige and Skoog medium supplemented with 9.2 μM zeatin. Best rooting rate of regenerated shoots was achieved on the same culture medium supplemented with 4.8 μM indole-3-butyric acid. In absence of morphological changes of in vitro regenerated plants acclimatized to the greenhouse, genetic conformity was assessed by simple sequence repeat screening. Our results suggest that in vitro propagation could be a useful tool for conservation of both diploid and triploid threatened Laperrine’s olive.     

In vitro propagation of Lilium testaceum and structural investigation of the storage β-1,4-glucomannan

Bulblet and callus cultures of Lilium testaceum were initiated in vitro from bulbscales. Continous propagation of the bulblet cultures was achieved on a modified Murashige and Skoog agar medium containing 1-naphthalene acetic acid (0.1 mg/l) and kinetin (0.1 mg/l) as phytohormones. The in vitro grown bulbs synthesized large quantities of storage ß-1,4-glucomannans (mannose: glucose = 73; molecular weight = 200 kd) with an identical structure to the glucomannans from the in vivo grown bulbs. Higher 1-naphthalene acetic acid concentrations (1 mg/l) resulted in increased callus formation. Liquid suspension cultures derived from callus exhibited only small amounts of reserve glucomannans.Abbreviations DEAE 2-(diethylamino)ethyl - GF growth factor - GM glucomannan - GPC gel permeation chromatography - IAA indole-3-acetic acid - IEC ion exchange chromatography - MS Murashige and Skoog - MW molecular weight - MWCO molecular weight cut off - NAA 1-naphthalene acetic acid - NMR nuclear magnetic resonance - PVP polyvinylpyrrolidone     

In vitro propagation and conservation of Satureja avromanica Maroofi—an indigenous threatened medicinal plant of Iran

Physiology and Molecular Biology of Plants - An efficient and rapid in vitro propagation system for Satureja avromanica, a rare and endangered folk medicinal plant of Iran was developed through the...