In vitro clonal propagation of Nyctanthes arbor-tristis

Rapid shoot multiplication of Nyctanthes arbor-tristis L. was achieved from axillary meristems on Murashige and Skoog (MS) basal medium supplemented with 1.0–1.5 mg dm⁻³ 6-benzylaminopurine (BA), 50 mg dm⁻³ adenine sulfate (Ads) and 3 % (m/v) sucrose. Inclusion of indole-3-acetic acid (IAA) in the culture medium along with BA + Ads promoted a higher rate of shoot multiplication. Maximum mean number of microshoots per explant (6.65) was achieved on the MS medium supplemented with 1.5 mg dm⁻³ BA, 50 mg dm⁻³ Ads and 0.1 mg dm⁻³ IAA after 4 weeks of culture. The elongated shoots rooted within 13 to 14 d on half-strength MS medium supplemented with either indole-3-butyric acid (IBA), IAA or 1-naphthaleneacetic acid (NAA) with 2 % sucrose. Maximum percentage of rooting was obtained on medium having 0.25 mg dm⁻³ IBA and 0.1 mg dm⁻³ IAA. About 70 % of the rooted plantlets survived in the greenhouse. The in vitro raised plants were grown normally in the field.     

Improved micropropagation of Gypsophila paniculata with bioreactor and factors affecting ex vitro rooting in microponic system

Studies on the mass production of high-quality plantlets in Gypsophila paniculata L. using a bioreactor and microponic system (a hydroponic system in which micropropagation shoots are planted) indicated that both aeration treatments, in which bioreactors were aerated from the top of explants by sparger (AS) and by tub (AT), were more effective than unaerated treatment for shoot proliferation and growth, and the maximum shoots (15.7 shoots per explant) with low hyperhydricity rate (2.9%) were found in the AS group. The ex vitro culture was more efficient for rooting when compared to the in vitro culture; the better shoot and root growth was obtained in the ex vitro culture, with rooting rate reaching 100% after 20 d of culture, but only 65% of in vitro shoots rooted; all stomata of ex vitro shoots closed, and their length was more than their width, but the stomata in in vitro shoots were all opened, the length close to the width. Furthermore, the stomata numbers were less in ex vitro (67.8) than in vitro (267.2). The survival rate of ex vitro plants reached 83.3% when plantlets derived in vitro and ex vitro were transferred to pots, while only 23.3% of in vitro plantlets survived. During ex vitro rooting with the microponic system, foam as the supporter material, 90 μmol?m^?2?s^?1 of light, and 80 shoots of planting density were favorable for shoot and root growth. The combination of bioreactor and microponic systems is an efficient way to produce high-quality plantlets of G. paniculata. Their application can reduce costs during large-scale industrial production.     

Factors affecting micropropagation and acclimatization of an elite clone of Eucalyptus tereticornis Sm.

Several factors influencing micropropagation of a selected elite clone of Eucalyptus tereticornis Sm. were investigated. Amongst different cytokinins tested, 6-benzyleadenine proved to be the most effective cytokinin for shoot multiplication and elongation. The initial size of the shoot clump (inoculum) also influenced shoot multiplication and elongation. The number of shoots proliferated per culture vessel were significantly higher (342 shoots per culture vessel) when larger shoot clumps (15?C20 shoots) were inoculated, compared to smaller shoot clumps (4?C5 shoots), which resulted in a reduced shoot proliferation rates (245 shoots per culture vessel). However, the number of elongated shoots (65 per culture vessel) and shoot length (5.23?cm) were higher in cultures which were inoculated with smaller shoot clumps in comparison to those cultures which were inoculated with larger shoot clumps (54 shoots per culture vessel with shoot length of 4.17?cm). The maximum number of rooted shoots (80.7?%) was obtained on one fourth-strength MS medium supplemented with 5.0???M indolebutyric acid. The number of shoots proliferated, elongated, rooting frequency, and subsequent survival of plants after acclimatization were higher in cultures incubated under photosynthetically active radiation (PAR) compared to those incubated under cool fluorescent lights (CFL). Osmotic potential of the sap and chlorophyll content of cultures incubated under PAR were also higher than those incubated under CFL. Following transfer of plants to soil, inoculation with a suspension of Bacillus subtilis (plant growth-promoting bacterium) increased the survival rate of plants by 10?%, yielding successful transfer of 84?% of plants. Random amplified polymorphic DNA and inter simple sequence repeat analyses indicated a high level of clonal uniformity amongst regenerated plants and also with that of the mother plant.     

Correction to: Genetic homogeneity assessment of in vitro-regenerated plantlets of Nyctanthes arbor-tristis L. and comparative evaluation of bioactive metabolites and antioxidant activity

In Vitro Cellular & Developmental Biology - Plant - There was an error in this article as originally published. The surname of coauthor Sobha Chandra Rath was misspelled as “Ratha”....     

In vitro micropropagation and ex vitro acclimation of Bupleurum kaoi-an endangered medicinal plant native to Taiwan

Summary This study reports an improved protocol for in vitro-shoot multiplication and ex vitro acclimation of Bupleurum kaoi, an endangered medicinal herb. Nodal segments were cultured in half-strength Murashige and Skoog (MS) basal medium supplemented with different concentrations of benzyladenine (BA) and kinetin. The presence of 0.25 mg l⁻¹ BA induced the highest number of shoots per explant after 8 wk of culture. Although BA was more effective than kinetin on shool multiplication, it induced hyperhydric shoots at all concentrations tested. The use of dispense paper (DP) instead of aluminum foil (AF) for container closure was found to reduce hyperhydricity and improve ex vitro acclimation. The best survival rate (61%) was obtained when plantlets were grown in MS basal medium containing 0.5 mg l⁻¹ indole-3-butyric acid and 0.1–0.2 mg l⁻¹ α-naphthaleneacetic acid using DP as container closure. Leaves of the plant treated with AF6 (two layers of AF as container closure and 6 wk of incubation) lacked epicuticular wax and possessed larger stomata, higher stomata density, and fewer functional stomata compared to those of plants treated with AF2+DP4 (two layers of AF for 2 wk, then replaced AF by three layers of DP for 4wk) and ex vitro-acclimated plantlets.     

Factors influencingin vitro micropropagation ofPinus strobus L.

Anin vitro procedure for micropropagation ofPinus strobus L., consisting of the following four steps has been established: shoot induction, shoot growth, root induction, and root growth. Influence of certain selected factors on each of these steps was determined. Shoot induction was found to be influenced by the age of the explant as well as the concentration of 6-benzyladenine in the medium. Best shoot growth was obtained on the medium of Litvayet al. without any growth regulators. Addition of activated charcoal to shoot growth medium resulted in fewer shoots. Root induction on several media was compared. Best root induction occurred when shoots were put on a half strength Gresshoff and Doy’s medium supplemented with 0.5 to 1.0 mg r1⁻¹ α-naphthylacetic acid (NAA) for two weeks. Shorter exposure of shoots to higher concentrations of NAA or indol-3-ylbutyric acid (IBA) was not as effective. Low temperature seemed to be a requirement for root growth.     

Wood ontogeny during ex vitro acclimatization in micropropagated hybrid poplar clones

Wood ontogeny patterns were determined during the ex vitro acclimatization period in micropropagated plantlets of hybrid poplar clones T-14 [Populus tremula × (Populus × canescens)] and T-50 [(Populus × canescens) × Populus tremula]. The temporal course of developmental changes in the woody tissue was characterized on a weekly basis starting from the day of transfer to the ex vitro environment until full acclimatization was achieved on day 28. In vitro rooted plantlets had already initiated lignification of secondary xylem cells. The greatest increase in the amount of woody tissue was observed on days 21 and 28. At the end of the acclimatization period, T-14 plantlets contained on average 41.4 % of secondary xylem tissue compared to 30.3 % found in T-50 plantlets. During the course of acclimatization, both clones displayed identical patterns of vessel lumen size distribution from small vessel lumen area to large vessel lumen area. This pattern differs from the characteristic diffuse-porous pattern of approximately evensized vessel lumen area distribution typical of mature wood. At the end of acclimatization, the differences in vessel lumen area and relative conductivity between the clones were negligible. Development of secondary xylem tissue during ex vitro acclimatization promotes the establishment of vigorous regenerants with stems that show increased bending strength and stiffness.     

Phenolic compounds profiles during ex vitro acclimatization of micropropagated Hypericum polyanthemum.

Accumulation of benzopyrans and total phenolic compounds were assessed in acclimatized field grown plants of Hypericum polyanthemum, an endemic species of southern Brazil, harvested at different developmental stages. The HPLC analysis of bioactive compounds 6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran (HP1), 7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethyl-benzopyran (HP2) and 5-hydroxy-6-isobutyryl-7-methoxy-2,2-dimethyl-benzopyran (HP3) revealed that the three benzopyrans are accumulated both in the vegetative and reproductive parts with maximum contents observed after 18 weeks (in the former) and 20 weeks (in the later) of plant growth (1.92+/-0.085 g % DW and 2.62+/-0.13 g % DW in the vegetative and reproductive parts, respectively). Highest contents of HP1 (1.56+/-0.12 g % DW) and HP2 (0.19+/-0.01 g % DW) were quantified in the green floral buds of the plants, whereas HP3 reached the highest level (1.02+/-0.08 g % DW) in the overblown flowers. The evaluation of total phenolic compounds showed that the vegetative parts accumulated the highest levels of the metabolites (51.93+/-0.67 mg QE (g DW)(-1)) after 16 weeks of plant growth. Considering the reproductive parts, the open flowers accumulated the greatest levels of the bioactive compounds (75.99+/-0.95 mg QE (g DW)(-1)). The results show that H. polyanthemum can be efficiently propagated and acclimatized to produce benzopyrans and other phenolic compounds.     

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.     

Meta-topolin and liquid medium mediated enhanced micropropagation via ex vitro rooting in Vanilla planifolia Jacks. ex Andrews

Plant Cell, Tissue and Organ Culture (PCTOC) - An advanced micropropagation protocol has been developed for the global spice crop Vanilla planifolia using meta-topolin [mT, 6-(3-hydroxybenzylamino)...     

Diversity and antimicrobial activity of endophytic fungi isolated from Nyctanthes arbor-tristis, a well-known medicinal plant of India

Endophytic fungi from Nyctanthes arbor-tristis were isolated and evaluated for their antimicrobial activity. A total of 19 endophytic fungi were isolated from 400 segments of healthy leaf and stem tissues of N. arbor-tristis. Eighteen endophytic fungi were obtained from leaf, while only ten from stem. Alternaria alternata had the highest colonization frequency (15.0%) in leaf, whereas Cladosporium cladosporioides ranked first in stem with a colonization frequency of 12%. The diversity and species richness were found higher in leaf tissues than in stem. The similarity indices between leaf and stem were 0.473 for Jaccard’s and 0.642 for the Sorenson index, respectively. Of 16, 12 (75%) endophytic fungal extracts showed antibacterial activity against either one or more pathogenic bacteria. The endophytic Nigrospora oryzae showed maximum inhibition against Shigella sp. and Pseudomonas aeruginosa. The leaf endophytes Colletotrichum dematium and Chaetomium globosum exhibited a broad range of anibacterial activity and were active against Shigella flexnii, Shigella boydii, Salmonella enteritidis, Salmonella paratyphi, and P. aeruginosa. Nine out of 16 (56.25%) endophytic fungi exhibited antifungal activity to one or more fungal pathogens. Colletotrichum dematium inhibited 55.87% of the radial growth of the phytopathogen Curvularia lunata. The antimicrobial activity of these endophytic microorganisms could be exploited in the biotechnological, medicinal, and agricultural industries.     

Factors affecting in vitro shoot proliferation and ex vitro establishment of mangosteen

Shoot proliferation has been achieved in Garcinia mangostana L. using seed explants. Maximum mean number of shoots per explant (16.8) was obtained from cultures on Murashige and Skoog medium supplemented with 40 mM 6- benzyladenine, and 2.5 mM -naphthaleneacetic acid and kept at 30 °C under an 8 hour photoperiod. Cultures on the same medium but supplemented with 2 g l^-1 activated charcoal produced fewer shoots. However, growth of these shoots was more organized and 75% rooting was obtained. Woody Plant Medium was not a suitable medium for shoot proliferation. Ex vitro establishment was best obtained on planting medium consisting of sand, soil and organic material (3:2:1).Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - MS Murashige & Skoog (1962) basal medium - WPM Woody Plant basal medium (Lloyd & Mc Cown 1980)     

Leucojum aestivum L. in vitro bulbs induction and acclimatization

The effects of growth retardants (paclobutrazol and ancymidol), sucrose, GA₃ (gibberellic acid) and physical state of the medium (solid and liquid — Rita® temporary immersion system) on in vitro induction of Leucojum aestivum bulbs and their acclimatization were studied. Paclobutrazol, regardless of the physical state of the medium, stimulated the formation of bulbs (99.3%). Under the influence 90 g L^?1 of sucrose or paclobutrazol the bulbs with the highest fresh weight (FW) were formed (250 mg and 208.8 mg, respectively). However, the addition of ancymidol to the liquid medium led to obtaining the bulbs showing the highest number of leaves and roots (63.2% and 91.7%, respectively). The scanning microscopy study proved that plants obtained in the medium containing GA₃ produced the stomata which most closely resembled to the one observed in the mother plant. Cytometric analysis of all regenerants revealed absence of changes in the nuclear DNA content. The maximum survival rate (100%) was observed for plants derived from liquid medium containing 90 g L^?1 of sucrose. Somewhat fewer plants were acclimatized after their cultivations in liquid medium enriched with paclobutrazol or ancymidol. The temporary immersion system led to perform successful ex vitro adaptation of Leucojum aestivum plants.     

Impact of sugar concentration in vitro on photosynthesis and carbon metabolism during ex vitro acclimatization of Spathiphyllum plantlets

Photosynthesis and carbon metabolism were followed during the acclimatization of micropropagated Spathiphyllum cv. Petite shootlets cultured with two different sucrose concentrations (3 and 6%). Increased sugar supply resulted in inhibition of photosynthesis, nonfunctional photosynthetic reaction centers, a more mixotrophic metabolism and higher starch and sucrose reserves at the end of the in vitro period. During the first days of acclimatization, net photosynthesis and adenosine diphosphoglucose (ADPG) pyrophosphorylase (EC 2.7.7.27) activity decreased for both treatments. In this period, sucrose was mainly used as nutrient reserve by plants cultured on 6% sucrose, as evidenced by a strong increase in sucrose synthase (EC 2.4.1.13) activity together with a severe decline in sucrose content. In the same period, sucrose breakdown and synthesis were in balance for plants cultured on 3% sucrose and the starch content increased slightly. After one week plants started to recover and full photosynthetic capacity developed. Two weeks after ex vitro transplantation, no differences between plants micropropagated with 3 or 6% sucrose could be found for photosynthesis, carbohydrate pools or enzyme activities. At the end of acclimatization the starch content increased again.     

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N⁶-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.     

Chlorophyll fluorescence as an indicator of photosynthetic functioning of in vitro grapevine and chestnut plantlets under ex vitro acclimatization

This study reports the effects of light availability during the acclimatization phase on photosynthetic characteristics of micropropagated plantlets of grapevine (Vitis vinifera L.) and of a chestnut hybrid (Castanea sativa × C. crenata). The plantlets were acclimatized for 4 weeks (grapevine) or 6 weeks (chestnut), under two irradiance treatments, 150 and 300 mol m^–2 s^–1 after in vitro phases at 50 mol m^–2 s^–1. For both treatments and both species, leaves formed during acclimatization (so-called `new leaves') showed higher photosynthetic capacity than the leaves formed in vitro either under heterotrophic or during acclimatization (so-called `persistent leaves'), although lower than leaves of young potted plants (so-called `greenhouse leaves'). In grapevine, unlike chestnut, net photosynthesis and biomass production increased significantly with increased light availability. Several parameters associated with chlorophyll a fluorescence indicated photoinhibition symptoms in chestnut leaves growing at 300 mol m^–2 s^–1. The results taken as a whole suggest that 300 mol m^–2 s^–1 is the upper threshold for acclimatization of chestnut although grapevine showed a better response than chestnut to an increase in light.     

Use of gentian violet to differentiate in vitro and ex vitro-formed roots during acclimatization of grapevine

The dye gentian violet was added to culture medium in order to distinguish in vitro and ex vitro-formed roots during acclimatization of micropropagated plantlets. Shoots of the grapevine rootstock Kober 5BB were rooted on media containing the dye (0.3 and 0.15 mg·l^–1) for 3 weeks. The dye coloured the roots. Root length was reduced by the presence of the dye, but root number and shoot growth were not affected. Most in vitro-formed roots continued to grow during acclimatization, and 3 weeks after the transfer to soil the root system was 60% composed of in vitro-formed roots. Our results suggest that in grapevine Kober 5BB, the in vitro-formed roots contribute to plantlet growth at least during acclimatization.     

Factors affecting efficient in vitro micropropagation of Muscari muscarimi Medikus using twin bulb scale

Endemic Muscari muscarimi Medikus is the most fragrant plant among Muscari species and has a high ornamental potential. The natural populations of M. muscarimi, are severely affected by increased environmental pollution and urbanization. There is a need to develop a micropropagation method that should serve effectively for commercial propagation and conservation. Therefore, the study targeted to set up a strategy for efficient in vitro bulblet regeneration system of M. muscarimi using twin scale bulb explants on 1.0 × MS medium containing 4.44, 8.88, 17.76 μM BAP (6-Benzylaminopurine) plus 2.685, 5.37, 10.74 μM NAA (α-Naphthalene acetic acid). Maximum number of 19 daughter axillary bulblets and 16 daughter adventitious bulblets per twin bulb scale explant was regenerated on 1.0 × MS medium containing 17.76 μM BAP plus 10.74 μM NAA and 17.76 μM BAP plus 2.685 μM NAA respectively. The daughter bulblets regenerated on twin bulb scales on 8 out of 9 regeneration treatment could be easily rooted on 1.0 × MS medium containing 4.9 μM IBA (Indole-3-butyric acid). The daughter bulblets regenerated on 9th treatment (1.0 × MS medium containing 17.76 μM BAP plus 10.74 μM NAA) were transferred to 1.0 × MS medium containing 30 g/l sucrose to break negative carry over effect of this dose of BAP–NAA, where they grew 2–3 roots of variable length. Daughter bulblet diameter was increased by culturing them on 1.0 × MS medium containing 4.44 μM BAP plus 5.37 μM NAA. The results verified that both age and the source of explants had significant effect on regeneration. In another set of experiments, twin scales were obtained from in vitro regenerated daughter bulblets, although they induced bulblets, yet their bulblet regeneration percentage, mean number of bulblets per explant and their diameter were significantly reduced. In vitro regenerated bulblets were acclimatized in growth chamber under ambient conditions of temperature and humidity on peat moss, where they flowered. The study provides important information about selection of suitable micropropagation medium, strategies to improve bulblet diameter and rooting of M. muscarimi which offers a scope for commercial propagation.Abbreviations: MS medium, Murashige Skoog medium; BAP, 6-Benzylaminopurine; NAA, α-Naphthalene acetic acid; IBA, Indole-3-butyric acid     

Mycorrhizal fungi enhancement of growth and gas exchange of micropropagated guava plantlets (Psidium guajava L.) during ex vitro acclimatization and plant establishment

Psidium guajava L.) plantlets was determined during acclimatization and plant establishment. Guava plantlets were asexually propagated through tissue culture and grown in a glasshouse for 18 weeks. Half of the plantlets were inoculated with a mixed endomycorrhiza isolate from Mexico, ZAC-19, containing Glomus diaphanum, G. albidum and G. claroides. Plantlets were fertilized with modified Long Ashton nutrient solution that supplied 11 μg P ml⁻¹. Gas exchange measurements were taken at 2, 4, 8, and 18 weeks after inoculation using a portable photosynthesis system. All micropropagated guava plantlets survived transplant shock. After 6 weeks, mycorrhizal plantlets had greater shoot growth rates and leaf production than non-mycorrhizal plantlets. This also corresponded with increased photosynthetic rates and stomatal conductance of mycorrhizal plants. By 18 weeks, mycorrhizal plantlets had greater shoot length, leaf area, leaf, stem, and root dry mass. However, gas exchange was comparable among treatments, in part because the container size was restricting growth of the larger mycorrhizal plantlets. Non-mycorrhizal plantlets had greater leaf area ratios and specific leaf areas than mycorrhizal plantlets. Increased leaf tissue mineral levels of P, Mg, Cu, and Mo also occurred with mycorrhizal plantlets. Roots of inoculated guava plantlets were heavily colonized with arbuscules, vesicles and endospores. Guava plantlets were highly mycotrophic with a mycorrhizal dependency index of 103%. Accepted: 27 December 1999