GIS-facilitated ex situ conservation of the rare Greek endemic Campanula incurva Aucher: Seed germination requirements and effect of growth regulators on in vitro proliferation and rooting

In order to develop conservation protocols for Campanula incurva, the geographical information systems (GIS) were used to unveil its ecological requirements; this facilitated the selection of substrates and of appropriate temperatures for cultivation and guided propagation experiments and acclimatization. Seed germination was tested under (i) dark, (ii) 16-h photoperiod, (iii) immersion in 400 ppm gibberellic acid (GA₃) followed by incubation at dark, and (iv) immersion in 400 ppm GA₃ followed by incubation at 16-h photoperiod (all at 21 ± 1°C). Dormancy was not detected. Germination exceeded 85% in 10 days. Shoot tips were established in vitro in Murashige and Skoog (MS) medium with 1 μM 6-benzyladenine (BA) and 0.1 μM indole-3-butyric acid (IBA). The effect of 1–8 μM BA and 1–8 μM kinetin on shoot proliferation was studied. Moreover, 8 μM BA was combined with 0, 1, 5, and 10 μM IBA to investigate effects of cytokinin/auxin. The highest number of microshoots/explant (4.03) was obtained with 8 μM BA. Microshoots were transferred to half strength MS and full strength MS media with 0, 0.5, 1, 5, and 10 μM IBA to evaluate their root induction ability. Half strength MS medium with 5 μM IBA resulted in 100% rooting (16.80 average number of roots/microshoot). Plantlets produced were successfully acclimatized.     

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.     

Micropropagation of Semecarpus anacardium L. from mature tree-derived nodal explants

A protocol was established for micropropagation of Semecarpus anacardium L. from mature tree-derived twigs. Sixty percent of aseptic cultures were obtained by surface sterilization with Bavistin, liquid detergent, and cefotaxime. Elongated twigs collected before flowering were optimum for in vitro culture initiation. Meristematic activity was triggered at all concentrations of thidiazuron (TDZ) incorporated into Woody Plant Medium. TDZ suppressed elongation of axillary buds, resulting into swollen meristems and upon its elimination multiple shoot primordia formation and differentiation were noted. Differentiation and shoot elongation were slower in explants pre-cultured with higher concentrations of TDZ. Swollen axillary meristems pre-cultured on TDZ (9.08 and 13.62 μM) failed to differentiate, whereas TDZ at 2.27 μM was optimal for shoot differentiation and elongation. Multiple bud induction was favored by 4.45 μM of TDZ. Differentiation of multiple shoot primordia by repeated subculturing on growth regulator-free medium and rooting was 100% in filter-paper supported half-strength liquid medium containing 7.38 μM IBA. Rooting was 90% in shoots placed directly in half-strength liquid medium with 2.46 μM IBA. Rooted plantlets hardened in soil:sand mixture (1:1) were transferred to green house. Genetic uniformity of in vitro raised clones with mother plant was confirmed by Inter-Simple Sequence Repeat markers.     

Micropropagation of <Emphasis Type="Italic">Metrosideros excelsa</Emphasis>

Multiple shoots were induced on stem segments of an 8-y-old plant of Metrosideros excelsa Sol ex Gaertn. “Parnel”. Axillary shoots produced on uncontaminated explants were excised, segmented, and recultured in the same medium to increase the stock of shoot cultures. The Murashige and Skoog (MS) medium, augmented with different concentrations of 2- isopenthenyladenine (2iP) and indole-3-acetic acid (IAA), either singly or in combinations, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment, equal molar concentrations of four cytokinins [2iP, kinetin, zeatin, and N ⁶-benzyladenine (BA)] in combination with equal molar concentrations of three auxins [IAA, α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA)] were tested for ability to induce axillary shoot development from single-node stem segments. The highest rate of axillary shoot proliferation was induced on MS agar medium supplemented with 1.96μM 2iP and 1.14μM IAA after 6 wk in culture. Different auxins (IAA, IBA, and NAA) were tested to determine the optimum conditions for in vitro rooting of microshoots. The best results were accomplished with IAA at 5.71μM (89% rooting) and with IBA at 2.85 or 5.71μM (86% and 86% rooting, respectively). Seventy and 90 percent of the microshoots were rooted ex vitro in bottom-heated bench (22 ± 2°C) after 2 and 4 wk, respectively. In vitro and ex vitro rooted plantlets were successfully established in soil.     

High-Frequency Regeneration by Abscisic Acid (ABA) from Petiole Callus of Jatropha curcas

Jatropha curcas L. is attaining worldwide interest as an important biofuel crop. Experiments were conducted to improve the prevailing micropropagation technique as well as to develop a new ex vitro rooting method for J. curcas plant regeneration. Regeneration and ex vitro rooting efficiency was enhanced by augmenting the culture medium with abscisic acid (ABA). Different concentrations of 6-benzylaminopurine (BAP) and indole-3-butyric acid (IBA) were tested for callus generation from both in vitro and in vivo explants (leaf and petiole) on Murashige and Skoog (MS) medium. The best regenerative callus was achieved on MS medium supplemented with BAP (4.44 μM) and IBA (2.45 μM) from in vitro-cultured petioles. Highest regeneration (91%) was achieved by culturing petiole callus on MS medium supplemented with BAP (8.88 μM), IBA (0.49 μM), and ABA (1.9 μM), whereas 61% regeneration was obtained from in vitro leaf callus. Shoot proliferation and elongation was achieved on BAP (2.22 μM) and IAA (8.56 μM) with 10–13 shoots per explants. Highest rooting (65%) was achieved from M1 shoots (BAP, IAA, and ABA) on MS medium supplemented with IBA (2.45 μM), naphthaleneacetic acid NAA (0.54 μM), and 0.02% activated charcoal. Ex vitro rooting of 1-mo-old M1 shoots obtained from the charcoal-containing medium resulted optimum rooting (>72%) when transferred to polybags containing sterile sand. The plantlets were successfully acclimatized in soil with more than 98% survival rate in the greenhouse.     

Influence of plant growth regulators on micropropagation and in vitro flowering of Trichodesma indicum (Linn) R. Br

An efficient protocol for micropropagation and in vitro flowering of Trichodesma indicum (Linn) R. Br. was developed using shoot tip explants. The physiological role of cytokinin and its combination with auxins on micropropagation and in vitro flowering was investigated. The highest number of shoots (9.94 ± 0.10) and the maximum average shoot length (5.56 ± 0.35 cm) were recorded on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP) (4.44 μM) and naphthaleneacetic acid (NAA) (2.69 μM). The effect of sucrose concentration on in vitro floral development was studied in plantlets cultured on MS medium supplemented with gibberellic acid (GA₃) and BAP. The highest percentage of flowering (93.2%) was obtained on MS medium supplemented with GA₃ (1.44 μM), BAP (1.33 μM) and sucrose (30 g l^?1). Root formation from the adventitious shoots was easily achieved on MS medium containing indole-3-butyric acid (IBA) (2.46 μM). The regenerated plantlets showed 86% survival rate and were phenotypically normal. The described method can be successfully employed for large-scale multiplication and in vitro flowering of T. indicum.     

Somatic embryogenesis and shoot regeneration from leaf derived callus of Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh

Abstract

The development of in vitro regeneration systems for Hypericum perforatum var. angustifolium (sin. Fröhlich) Borkh, a medicinal plant used for treating neurological disorders, is described. For the first time in this variety, somatic embryogenesis and shoot regeneration were induced from leaf-derived callus. Well-formed plantlets were obtained through both shoot regeneration and somatic embryogenesis, with separate morphogenetic programmes. Proembryogenic masses were obtained in liquid MS and B5 media supplemented with 5.8 μM 2,4-D, 1.34 μM NAA, and 1.16 μM Kin; after being transferred onto hormone-free medium, they formed whitish and spherical structures that subsequently developed into the heart and torpedo stages.

On MS agarized medium containing thidiazuron (TDZ) at different concentrations (3, 6, 9, 12 μM) combined with 2 μM IBA, only shoot regeneration, and not somatic embryogenesis, was obtained. The mean number of shoots increased significantly when the concentration of TDZ was 3 μM.     

Nutrient alginate encapsulation of nodal segments of Althaea officinalis L., for short-term conservation and germplasm exchange

Abstract

In the present study, an alternate method for germplasm storage in the form of artificial seeds was standardized via nodal explants excised from in vitro proliferated shoots. The explants were encapsulated using sodium alginate and calcium chloride as gelling matrix. For development of root along with shoot, excised nodal segments were pretreated with ½ MS medium along with 20 μM IBA for 24 h and encapsulation was carried thereafter. Combination of 3% sodium alginate augmented with 100 mM CaCl₂.2H₂O was found appropriate for the formation of clear and uniform beads and subsequent conversion of encapsulated nodal segments into plantlets. Maximum (66%) encapsulated nodal segments were converted into plantlets on MS medium supplemented with 7.5 μM BA and 0.5 μM NAA after eight weeks. Regeneration frequency of auxin-pretreated encapsulated and non-encapsulated nodal segments (stored at 4 ºC) was evaluated at different storage time (0 to 6 weeks). After four weeks of storage, encapsulated propagules exhibited highest conversion response on the optimized medium after eight weeks of culture. Plantlets were hardened and established with success in ex vitro conditions. Conversion of synthetic seeds into plantlets was observed when these were directly sown in autoclaved Soilrite^TM (Keltech Energies, Bangalore, India).     

Potential role of cytokinin–auxin synergism, antioxidant enzymes activities and appraisal of genetic stability in Dianthus caryophyllus L.—an important cut flower crop

The regeneration potential, antioxidative enzyme activities, and genetic stability among micropropagated plantlets of Dianthus caryophyllus L. were evaluated. Multiple adventitious shoots were induced from leaf explants on Murashige and Skoog medium incorporated with various combinations and concentrations of plant growth regulators (PGRs). The highest leaf explant response (90%), number of shoots per explant (15.30?±?1.19), and shoot length (6.75?±?0.63 cm) was recorded in response to a combination of 2.5 μM 6-benzyladenine and 0.5 μM α-naphthaleneacetic acid (NAA) after 8 wks culture. Subsequent subculturing for five passages, on a medium with the same composition of PGRs, induced the highest shoot number (42.50?±?1.44), with an average shoot length of 8.06 cm after the fourth subculture. Different concentrations of indole-3-butyric acid (IBA) were tested to determine the optimum conditions for ex vitro rooting of microshoots. The best result was accomplished with a pulse treatment of IBA (100 μM) applied to the basal end of the microshoot for 30 min, followed by transfer to plastic cups containing soilrite, and eventually established in natural soil with an 85% survival rate. The determination of activities of antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, catalase, and glutathione reductase) revealed involvement of these enzymes in shoot differentiation and development. All of these activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. Intersimple sequence repeat DNA analysis was carried out using five primers. The amplification products were monomorphic in micropropagated plants, similar to those of the mother plant. No polymorphisms were detected revealing the genetic integrity of the micropropagated plants.     

Rapid in vitro propagation of Eclipta alba (L.) Hassk. through high frequency axillary shoot proliferation

An efficient protocol has been developed for rapid in vitro propagation of Eclipta alba L. (Asteraceae) through axillary bud multiplication. Murashige and Skoog (MS) medium supplemented with BA (10 M) was found to be most effective in breaking bud dormancy. An average number of 23 ± 0.57 shoots per explant was recorded after 30 days. Culture of node segments on fresh medium with lower concentration of BA (2 M) enhanced the multiplication rate. A maximum of 79 ± 1.90 mean number of shoots were obtained after three subcultures without any decline in multiplication rate. The regenerated microshoots showed the most efficient rooting on half strength MS medium augmented with 0.5 M IBA. Plantlets went through a hardening phase prior to ex vitro transfer and established in earthen pots containing garden soil; survival of about 90%. The established plants were uniform and exhibited morphological characters identical to mother plants.     

Direct shoot organogenesis from hypocotyl explants of Jatropha curcas L. an important bioenergy feedstock

Multiple shoots were induced on hypocotyl segments reared from in vitro germinated seedlings of Jatropha curcas L., using Murashige and Skoog (MS) medium supplemented with N⁶‐benzyl adenine (BA) and kinetin (kin) either alone or in combination with indole‐3‐acetic acid (IAA). The combined treatment of 7.05 μm kin and 1.425 μm IAA resulted in maximum shoot production with an average shoot bud initiation of 12.1 per explant. The regenerated microshoots were transferred to root induction medium containing half‐strength MS salts supplemented with either indole‐3‐butyric acid (IBA) or IAA. Rooting of microshoots was best achieved on half‐strength MS medium supplemented with IBA (9.8 μm ). Rooted plantlets were subsequently acclimatized under green house condition and the plantlets showed 70% survival.     

Rapid plant regeneration and analysis of genetic fidelity in micropropagated plants of Vitex trifolia: an important medicinal plant

An efficient in vitro regeneration protocol of a valuable medicinal plant, Vitex trifolia has been successfully established using nodal segments as explants. Three different cytokinins (BA, Kn, 2iP) and auxins (NAA, IAA, IBA) in different concentrations and combinations, evaluated as supplements to Murashige and Skoog’s medium showed to have a marked influence on the regeneration output. Among all the single cytokinin treatments MS medium supplemented with 5.0 μM BA produced the maximum number of shoots yielding 8.20 ± 0.37 shoots per explant with 4.8 ± 0.43 cm shoot length after 8 weeks of culture. Combined with low auxin concentrations, all the three cytokinins at their optimal concentrations synergistically enhanced the regeneration credentials. However, MS medium enriched with 5.0 μM BA and 0.5 μM NAA yielded the best possible regeneration in the species with a regeneration percentage of 97.33 ± 2.67 % and amounting to 16.80 ± 0.58 shoots per explant with 6.20 ± 0.25 cm mean shoot length at the end of 8 weeks in culture. Ex vitro rooting of in vitro derived microshoots was achieved by 20 min 500 μM IBA treatment followed by transfer to thermocol cups containing sterile soilrite. A 95 % plantlets survived acclimatization procedure to the field. Genetic conformity of the regenerated plants was established through RAPD. All the bands visualized on agarose gels were monomorphic with that of the donor plant indicating the clonal nature of the regenerants.     

Effect of N6-isopentenyladenine concentration on growth initiation in vitro and rooting of bilberry and lingonberry microshoots

Buds and shoot tips of wild bilberry (Vaccinium myrtillus L.) and lingonberry (V. vitis-idaea L.) plants were cultured on a modified MS medium containing N⁶-isopentenyladenine (2iP), 9.8–78.4 μM, in order to study the effect of the 2iP-concentration on the initiation of growth. The experiment was first performed in the autumn and repeated in the spring to determine the influence of season on growth initiation. To optimise rooting, three different rooting treatments were tested for the bilberry and lingonberry microshoots. Shoots were rooted either in vitro with 0.49 μM IBA (indole-3-butyric acid) or ex vitro, incubating microshoots in 2.07 mM KIBA-solution (potassium salt of IBA) before planting, or microshoots were planted directly on peat without exogenous auxin. The best 2iP concentration for the initiation of the growth for bilberry was 49.2 μM and for lingonberry 24.6 μM. It was observed that increasing the 2iP concentration at the growth initiation stage increased the number of brownish explants both in bilberry and in lingonberry microcultures. Spring was a considerably better time than autumn for the initiation of new growth, for both species. The results of the rooting test showed that the KIBA-treatment before planting on peat increases rooting efficiency in both bilberry and lingonberry. This revised version was published online in June 2006 with corrections to the Cover Date.     

PLANT GROWTH REGULATORS ENHANCE GOLD UPTAKE IN BRASSICA JUNCEA

The use of plant growth regulators is well established and they are used in many fields of plant science for enhancing growth. Brassica juncea plants were treated with 2.5, 5.0 and 7.5 μM auxin indole-3-butyric acid (IBA), which promotes rooting. The IBA-treated plants were also sprayed with 100 μM gibberellic acid (GA₃) and kinetin (Kin) to increase leaf-foliage. Gold (I) chloride (AuCl) was added to the growth medium of plants to achieve required gold concentration. The solubilizing agent ammonium thiocyanate (1 g kg^?1) (commonly used in mining industries to solubilize gold) was added to the nutrient solution after six weeks of growth and, two weeks later, plants were harvested. Plant growth regulators improved shoot and root dry biomass of B. juncea plants. Inductively Coupled Plasma Optical Emission Spectrometry analysis showed the highest Au uptake for plants treated with 5.0 μM IBA. The average recovery of Au with this treatment was significantly greater than the control treatment by 45.8 mg kg^?1 (155.7%). The other IBA concentrations (2.5 and 7.5 μM) also showed a significant increase in Au uptake compared to the control plants by 14.7 mg kg^?1 (50%) and 42.5 mg kg^?1 (144.5%) respectively. A similar trend of Au accumulation was recorded in the roots of B. juncea plants. This study conducted in solution culture suggests that plant growth regulators can play a significant role in improving phytoextraction of Au.     

Micropropagation of Pistachio from mature trees

Multiple shoots were induced from nodal segments of mature trees of Pistacia vera L. on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BA). Maximum shoot production was obtained from shoot tips taken from in vitro proliferated shoots when cultured on solidified MS medium containing 8.8 μM BA. The multiplication rate was 20 microshoots per explant on the 30th day. Rooting of microshoots was achieved in MS medium supplemented with indole butyric acid (IBA). Rooted plantlets reassumed independent growth after a short period of acclimatisation. Stable regenerated plants were established in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation and salt tolerance of in vitro grown Crithmum maritimum L.

Crithmum maritimum (Apiaceae), a perennial halophyte native in Greece, could be used as an alternative culture at problematic soils. It presents significant economical potentials as its essential oils are in high demand from the medicinal and cosmetic industry. The response of the species on in vitro conditions was studied. MS proved to be the most effective of the basal media tested for in vitro adventitious shoot production, resulting in significantly increased number of new microshoots/explant and higher shoots. 6-Benzyladenine (BA) at 2.5 μM increased shoot proliferation. The combination of α-Naphthaleneacetic acid (NAA) (1–2.5 μM) with BA (2.5 μM) had a positive influence at simultaneous proliferation and rooting resulting in high rooting percentage (82.5–95%) and increased number of roots. Rooting percentage reached 100% and number of roots increased significantly when 0.5 μM and 1 μM IBA was combined with ½MS and full strength MS. The in vitro response to salinity stress (0–300 mM NaCl) was also tested. Shoot proliferation was gradually reduced at higher concentrations of NaCl but shoot height was enhanced. Acclimatization procedure was successful.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Artemisia abrotanum</Emphasis> L. by means of somatic organogenesis

The goal of this project was to regenerate Artemisia abrotanum L., Southern wormwood, by means of organogenesis from leaves. In vitro plant propagation may greatly support the molecular characterization of the medicinal qualities of A. abrotanum. Young, intact leaves were excised from mature plants and surface sterilized. Abundant callus growth, as well as shoot formation, was produced on an MS medium supplemented with 4.44 μM BA and 0.54 μM or 0.81 μM NAA. Shoots, with some residual callus, rooted equally well in MS media with 0.49 μM IBA, 0.54 μM NAA, or without hormones. Rooted plants were best acclimated in potting soil.     

Influence of auxins and darkness on in vitro rooting of micropropagated shoots from mature and juvenile Acacia mangium

The influence of total darkness versus a 16/8 photoperiod and of auxins added to the culture medium on the in vitro root formation capacity of Acacia mangium microshoots of juvenile and mature origin was examined. Rooting of the mature clone was significantly increased by exposing the microshoots to auxins (4 and 6 μM IAA or IBA) in darkness, while the promoting effect of darkness combined with 4 μM IAA was more time-restricted for the juvenile-origin microshoots. Overall, the latter rooted in greater proportions than those from the mature source. Maintaining the microshoots of both origins on auxin supplemented medium in darkness resulted in a greater number of adventitious roots formed than under the standard 16/8 lighting conditions. On the other hand, light stimulated root elongation. These results are discussed mainly from the viewpoint of auxin metabolism in relation to adventitious root formation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Encapsulation of nodal segments of Cassia angustifolia Vahl. for short-term storage and germplasm exchange

The present study described the encapsulation of nodal segments of Cassia angustifolia Vahl. excised from 1-month-old in vitro raised cultures for short-term conservation and propagation. Various concentrations and combinations of gelling matrix (sodium alginate) and complexing agents (calcium chloride) were tested to prepare uniform beads. The ideal beads were obtained through a combination of 3 % sodium alginate and 100 mM calcium chloride. The maximum conversion response (94 %) of encapsulated beads was obtained in Murashige and Skoog’s medium (MS medium) supplemented with 2.5 μM benzyladenine (BA) and 0.4 μM α-naphthalene acetic acid (NAA) after 6 weeks of culture. The encapsulated and non-encapsulated nodal segments were also stored at 4 °C for different time periods (0, 1, 2, 4, 6 and 8 weeks). The regenerated microshoots were best rooted in optimized rooting medium that comprised half-strength MS + 1.0 μM indole-3-butyric acid (IBA) + 5.0 μM phloroglucinol (PG) for the production of complete plantlets. The regenerated plantlets were successfully hardened and acclimatized in natural conditions with 70 % survival rate.     

Micropropagation of Telopea speciosissima R. Br. (Proteaceae). 2: Rhizogenesis and acclimatisation to ex vitro conditions

Conditions affecting rhizogenesis in vitro and ex vitro and subsequent acclimatisation of Telopea speciosissima (waratah) were investigated. Clonal selections were successfully rooted in vitro in agar, on filter paper bridges or using crushed quartz-sand, the last substrate resulting in superior growth of roots. The in vitro substrates were impregnated with half-strength MS, 7.5 gl^-1 sucrose and various concentrations of IBA. For the quartz-sand, an IBA concentration of 50 M was optimal, 70% of microcuttings were rooted. No plantlets rooted in vitro were acclimatised to ex vitro conditions (using mist, fog or humidity tent regimes). Microcuttings (25–45 mm in length) were rooted ex vitro in a fog humidity regime (droplet size <10 m) using an IBA powder dip (3 g IBA kg^-1). Neither a mist nor a humidity-tent regime was suitable for rooting of waratah microshoots ex vitro. A peat and perlite mixture was superior to crushed quartz-sand or potting mix for the rooting of microshoots; this appeared to be related to the air-filled porosity (>20%) of the mixture, measured after the medium was saturated and then drained for 24h. Plantlets must be left under the high humidity regime until shoot growth resumes (four to eight weeks) otherwise plant mortality increase significantly. In vitro-produced leaves abscised between eight and 12 weeks after transfer to ex vitro conditions, indicating that these structures did not acclimatise ex vitro.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IBA indole-3-butyric acid - LSD least significant difference - MS Murashige and Skoog medium