In vitro plantlets from alginate-encapsulated shoot tips of Solanum nigrum L.

Shoot tip explants obtained from in vitro proliferated shoots were encapsulated in 3% sodium alginate and 100 mM calcium chloride for the production of synthetic seed in Solanum nigrum L., a medicinally important plant. Morphogenic responses of encapsulated shoot tips to various sowing media (full or half-strength 0.8% agar-solidified or liquid MS medium or full-strength MS medium containing BAP) were evaluated in vitro. Of the six media evaluated, maximum conversion was obtained on 0.8% agar-solidified growth regulator free full-strength MS medium. The addition of MS nutrients in alginate matrix had a pronounced effect on the length of shoots that emerged from alginate beads. Encapsulated shoot tips also converted when directly sown in sterile soil moistened with liquid MS medium. Encapsulated shoot tips could be stored at low temperature (4 °C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully.     

Cryopreservation of carnation (Dianthus caryophyllus L.) shoot tips by encapsulation-vitrification

Shoot tips excised from in vitro cultured plants of Dianthus caryophyllus L. (cv. Pallas, cv. Pink Candy and cv. Wanessa) were successfully cryopreserved using an encapsulation-vitrification method. Shoot tips (2–3 mm in length) were encapsulated in sodium alginate, precultured on liquid Murashige and Skoog (1962) medium supplemented with various sucrose concentrations (0.25, 0.5, 0.75, 1.0 M) for 24 h or 48 h and dehydrated with the vitrification solution PVS2 (up to 4 h) at 24 °C or 0 °C prior to direct immersion in liquid nitrogen (−196 °C). A maximum of shoot regeneration from cryopreserved shoot tips was obtained with the following combinations: preculture in 0.5 M sucrose and 180 min dehydration treatment at 0 °C for cv. Pallas (60% shoot formation), or preculture in 0.75 M and 200 min dehydration at the same temperature for cv. Pink Candy (66.6% shoot formation) and cv. Wanessa (73% shoot formation).     

In vitro proliferation and minimum growth storage of fraser photinia: Influences of different medium,sugar combinations and culture vessels

Protocols for the in vitro proliferation and storage of fraser photinia were developed by comparing 6-benzyladenine (BA) concentrations (0.5–4 mg/L) together with different media formulations [Murashige and Skoog (MS) media and Quoirin and Lepoivre (QL) media], sugar combinations (sucrose and mannitol), culture vessels (baby food jars and vitrovents) and methods (synthetic seed technology and slow growth storage). The best responses in terms of both proliferation percentage and multiple shoot formation were obtained in QL medium containing 1 mg/L BA. Synthetic seed production was optimized by encapsulating shoot apices in 3% sodium alginate. Encapsulated shoot apices could be maintained up to 6 months at 4 °C in dark with 91.6% sprouting in MS medium. Microshoots were stored at 4 °C up to 15 months on sucrose and mannitol containing QL medium in both baby food jars and vitrovents without subculture. The stored material could be recovered and multiplied normally in 1 mg/L BA supplemented QL medium. Both in vitro propagated and conserved microshoots were rooted (∼75%) on QL medium with 1 mg/L indole butyric acid (IBA). Optimized synthetic seed and slow growth storage system can be used for short and medium-term storage of fraser photinia germplasm.     

Nutrient-alginate encapsulation of in vitro nodal segments of pomegranate (Punica granatum L.) for germplasm distribution and exchange

The present paper demonstrates the potential of nutrient-alginate encapsulation of axenic nodal segments of pomegranate for synthetic seed technology, which could be useful in germplasm distribution and exchange. Nodal segments from in vitro shoot cultures derived from mature nodal explants (source A) or axenic cotyledonary nodes (source B) were encapsulated in calcium alginate hydrogel containing Murashige and Skoog's [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497] medium (MS) supplemented with 4.44 μM benzyladenine (BA) and 0.54 μM naphthalene acetic acid (NAA). Of various concentrations of sodium alginate (1–6%) and the complexation solution of calcium chloride (50–125 mM), a combination of 3% sodium alginate and 100 mM calcium chloride was most suitable for formation of ideal synthetic seeds. Morphogenic response of encapsulated nodal segments to seven different planting media was evaluated. Encapsulated nodal segments of both the sources exhibited shoot development only in four selected media. Of the planting media evaluated, % sprouting (shoot development) was the highest in MS medium augmented with 4.44 μM BA and 0.54 μM NAA and lowest in (1/2) MSS medium. One step germination i.e. both shoot and root formation was possible only with encapsulated nodal segments of source B in MS, (1/2) MSS and natural soil + (1/2) MSS, with MS being most effective. Encapsulated nodal segments stored up to 30 days at 4 °C were capable of sprouting. Plants regenerated from the encapsulated nodal segments were hardened off and transferred to soil.     

Cryopreservation of shoot apices of hawthorn in vitro cultures originating from East Asia

The objective of this study was to establish a cryopreservation protocol for hawthorn shoot apices (Crataegus pinnatifida Bge.). Cryopreservation was carried out via encapsulation–dehydration, vitrification, and encapsulation–vitrification on shoot apices excised from in vitro cultures. We began by showing that cold-acclimation enhanced the regrowth of cryopreserved apices from 10.0 to 65.5% in encapsulation–dehydration. We then decided that the encapsulation–dehydration method was an optimal cryopreservation method for hawthorn shoot apices in terms of its high recovery after cryopreservation as well as its ease of use compared with vitrification and encapsulation–vitrification. In encapsulation–dehydration, the protocol leading to optimal regrowth was as follows: after cold-acclimation at 5 °C in the dark for 2 weeks, excised shoot tips were pretreated for 24 h at 25 °C on hormone-free Murashige and Skoog [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497] (MS) basal medium with 0.4 mol/L sucrose, then encapsulated and precultured in liquid MS medium with 0.8 mol/L sucrose for 16 h at 25 °C. Precultured beads were dehydrated for 6 h at 25 °C in the dessicator containing 50 g silica gel to a moisture content of 15.3% (fresh-weight basis) before cryostorage for 1 h. In addition, we examined the effect of adding glycerol to both the alginate beads and loading solution to enhance regrowth after cryopreservation in encapsulation–dehydration. In the present study, it was shown that adding 0.5 mol/L glycerol resulted in high regrowth percentages (82.5–90.0%) in four Crataegus species.     

Encapsulation for in vitro short-term storage and exchange of ginger (Zingiber officinale Rosc.) germplasm

Synseeds of ginger (Zingiber officinale) were produced using aseptically proliferated 2-week old encapsulating explants (microshoots) upon complexation of 4% sodium alginate prepared in Murashige and Skoog (1962) medium (MS) and 100 mM calcium chloride. Conversion of synseeds into plantlets (conversion) was recorded as 66% and 53% on MS (3% sucrose) and on MS (3% sucrose) + 2.5 mg/l BA media, respectively. However, shoots/synseed were significantly higher on MS (3% sucrose) + 2.5 mg/l BA medium. For short-term storage of germplasm, sucrose-dehydrated synseeds were found better than air-dehydrated or fresh synseeds. Synseeds dehydrated in 0.25 M sucrose liquid medium for 16 h and stored in cryovials (with out medium) at 25 °C for 8 weeks and 12 weeks exhibited 53% and 13% conversion, respectively, on MS (3% sucrose) + 2.5 mg/l BA medium. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plants. This synseed protocol could be useful for short-term storage and exchange of germplasm of ginger between national as well as international laboratories.     

Effect of ABA and sucrose on germination of encapsulated somatic embryos of guava (Psidium guajava L.)

Present study demonstrates the effect of sucrose and ABA on germination of encapsulated somatic embryos of guava (Psidium guajava L.). Sucrose and ABA at different concentrations were also evaluated for their effects on maturation and germination of somatic embryos. Mature somatic embryos developed on MS medium containing high concentration of sucrose (10%) or ABA (1.0 mg l⁻¹) showed inhibition in germination if they continued to be in same medium for 4 weeks. With increasing concentrations of sucrose (3–9%) or ABA (0.01–1.0 mg l⁻¹) in medium, percent germination of encapsulated somatic embryos decreased significantly. Encapsulated somatic embryos after storage on MS medium supplemented with 9% sucrose or 1 mg l⁻¹ ABA for different duration (0–60 days) germinated when they were transferred to medium containing 3% sucrose. About 20.8% and 37.5% encapsulated somatic embryos germinated after storage on ABA (1 mg l⁻¹) or sucrose (9%) for 60 days, respectively. Temporarily suppression in germination of encapsulated somatic embryos by high concentration of sucrose or ABA may be important for short-term conservation of elite genotype of guava.     

Droplet-vitrification of apical shoot tips of Rubus fruticosus L. and Prunus cerasifera Ehrh.

Apical shoot tips excised from in vitro plantlets of blackberry (Rubus fruticosus L. ‘?a?anska Bestrna’) and cherry plum (Prunus cerasifera Ehrh.) were tested for recovery after cryopreservation using the droplet-vitrification technique. Following treatment for 30 min with a loading solution comprising 1.9 M glycerol and 0.5 M sucrose, explants were dehydrated with a highly concentrated cryoprotectant solution, so called vitrification solution. Shoot tips were dehydrated for 10, 20 and 30 min at room temperature with a solution derived from the original PVS2 solution (containing 37.5% (w/v) glycerol, 15% (w/v) dimethylsulfoxide, 15% (w/v) ethylene glycol and 22.5% (w/v) sucrose) and for 60, 90 and 120 min using the PVS3 solution (containing 50% (w/v) glycerol and 50% (w/v) sucrose). Explants were cooled by direct immersion in LN in 10 μl droplets of vitrification solution placed on aluminium foil strips. Rewarming was done by direct plunging of foil strips in a preheated (37 °C) unloading solution (0.8 M sucrose) for 30 s, after which an equal volume of unloading solution (at room temperature) was added for further incubation for 30 min. As for regrowth of blackberry, PVS3 proved more effective than the modified PVS2, but the difference was significant (P < 0.05) only for the shortest treatment duration. The duration of PVS3 treatment had no significant effect on regrowth of cryopreserved shoot tips (45.8–70%). By contrast, a 30-min treatment with modified PVS2 solution resulted in a significant increase in regeneration percentage (30%), as compared with a 10-min treatment with the same solution (5%). Cherry plum shoot tips were very sensitive to both vitrification solutions and growth recovery of cryopreserved samples was generally lower (5–20%) than that of blackberry explants. No significant influence of PVS treatment (both type of solution and treatment duration) on regrowth of cryopreserved shoot tips was observed with cherry plum shoot tips. Experiments performed in France and in Serbia produced similar results, thereby showing the robustness and reproducibility of the protocols developed.     

Cryopreservation of Limonium serotinum apical meristems from in vitro plantlets using droplet-vitrification

In this study in vitro shoot tips of a Sicilian genotype of Limonium serotinum were successfully cryopreserved using the droplet-vitrification technique. Growth recovery of cryopreserved shoot tips was possible only when samples were pretreated for 16 h in liquid medium with 0.3 M sucrose, then for 5 h in liquid medium with 0.7 M sucrose before performing the cryopreservation protocol. Optimal conditions included treatment for 20 min in a loading solution containing 1.9 M glycerol + 0.5 M sucrose, treatment with vitrification solution B5 (glycerol 40.0%, sucrose 40.0%, w/v) for 60 and 90 min or vitrification solution A9 (glycerol 30.0%, dimethylsulfoxide 20.0%, ethylene glycol 20.0%, sucrose 15.0%) for 20 min, rapid cooling in minute droplets of vitrification solution, rapid rewarming by immersion for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 37% recovery of cryopreserved shoot tips was achieved. Regrowth of cryopreserved samples was slow but always direct, without callus formation.     

High frequency plantlet regeneration from callus and artificial seed production of rock plant Pogonatherum paniceum (Lam.) Hack. (Poaceae)

Pogonatherum paniceum (Lam.) Hack. is a rock plant with good potential for vegetative recovery on naked lands. A high frequency in vitro regeneration system was developed for P. paniceum. Calli were induced from explants of mature seeds, seedlings, young leaves, and stem segments on Murashige and Skoog (MS) medium supplemented with 1.0 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 2.0 mg L⁻¹ α-naphthalene acetic acid (NAA) and 0.2 mg L⁻¹ 6-benzylaminopurine (BAP). High induction rates (59.57%) and regeneration rates (100%) were obtained from mature seed explants; calli were sub-cultured for over 2 years and still retained a high regenerative capacity. One seed explant resulted in 69,997 plants in 1 year. Shoot buds derived from calli were used for encapsulation in liquid MS medium containing 3% sucrose and two different alginate matrices (3% sodium alginate (w/v) + MS medium containing 3% sucrose and 3% sodium alginate + 1% activated carbon (w/v) + MS medium containing 3% sucrose) with a 20-min exposure to 2% CaCl₂ and 0.3% bavistin (w/v). The capsule with 3.0% sodium alginate (w/v) and 1% activated carbon (w/v) showed a higher conversion rate (61.58%) and stronger plantlets under non-aseptic conditions. These systems are useful for the rapid clonal propagation and dissemination of artificial seed material of P. paniceum for eco-recovery.     

Regeneration of Plants from Alginate-encapsulated Shoot Tips of Myrtle (Myrtus communis L.)

Myrtle growing naturally in the Mediterranean Region in Turkey, is among the economically important plant species. The present study emphasized on in vitro conservation of M. communis by encapsulating regenerated shoot tips. In this research we reported synthetic seed production and subsequent conversion of encapsulated shoot tips into plantlets comparing with nonencapsulated shoot tips for myrtle. Two different myrtle genotypes were used for synthetic seed production. Sodium alginate solution at the rate of 3.0% and 100?mM calcium chloride solution were prepared for encapsulation. Encapsulation was accomplished by mixing shoot tips into sodium alginate solution and dropping these in calcium chloride solution for 25–30?min. Encapsulated and nonencapsulated shoot tips were cultured in MS (Murashige and Skoog) media supplemented with different BAP (6-Benzylaminopurine) concentrations (0, 0.5, 1, 2?mg L^?1). After six weeks, all shoots were transferred to MS media containing 1?mg L^?1 IBA for in vitro rooting. As a result, the highest germination rate was obtained on the BAP-free MS media. The best BAP concentrations were detected as 0.5 and 1?mg L^?1 for micropropagation. Genetic stability of plants coming from encapsulated and nonencapsulated shoot tips was tested by ISSR markers. Based on the results, there were no genetic differences among the samples.

     

Storage and high conversion frequency of encapsulated protocorm-like bodies of Cymbidium devonianum (orchid)

Summary

A method for the storage and high frequency conversion of Cymbidium devonianum protocorm-like bodies (PLBs) is reported. To study the effect of nutrient level on storage, PLBs were encapsulated in calcium alginate beads supplemented with 1.0×, 0.5×, 0.25×, or 0.125× Murashige and Skoog (MS) basal medium containing 0.3% (w/v) sucrose, without agar in the encapsulating matrix, and stored at room temperature (25º ± 2ºC) in the dark. Beads containing 0.25× MS were also kept at different temperatures (0ºC, 4ºC, 8ºC, or room temperature) in the dark to ascertain the optimal temperature for storage. One set of controls (i.e., non-encapsulated PLBs) was maintained for each treatment. The survival and subsequent percentage conversion values of PLBs were assessed at 30 d storage intervals after culturing on MS regeneration medium. Compared to the controls, all encapsulated PLBs showed improved storage at room temperature. In all treatments, non-encapsulated PLBs did not survive, but turned brown and died. Encapsulated PLBs in 0.25× MS medium could be stored for 90 d at room temperature without any significant loss in viability. However, a significant decrease in the survival percentage was recorded after longer storage times. Encapsulated PLBs containing 0.25× medium could be stored at 4ºC and 8ºC for 120 d and 180 d, respectively, without loss of viability. As storage times increased beyond 180 d, the survival percentage of encapsulated PLBs decreased. The conversion of encapsulated PLBs led to the emergence of regenerated plantlets. Initially, small green globular outgrowths from the PLBs were observed on the surface of the beads. These outgrowths multiplied to form clusters of PLBs which then regenerated into plantlets. In both studies, more prolonged storage of encapsulated PLBs increased the time interval for germination and plantlet regeneration on MS regeneration medium. Similarly, decreases in both basal MS strength in the matrix, and in storage temperature, resulted in an increase in the time required for germination and plantlet regeneration. Plantlets that regenerated from stored, encapsulated PLBs were hardened-off, and a high survival percentage (90%) was obtained in a glasshouse.     

Production of protocorm-like bodies with bioreactor and regeneration in vitro of Oncidium ‘Sugar Sweet’

To produce mass propagules of Oncidium ‘Sugar Sweet’, we tested the feasibility of producing protocorm-like bodies (PLBs) using 5 l balloon type air-lift bioreactors, and selected an optimal culture medium for shooting and rooting in vitro. The results showed that liquid bioreactor cultures were more efficient for PLBs proliferation when compared to solid- and liquid-agitated flask cultures. The maximum PLBs biomass (326.3 g per bioreactor, FW) was obtained in the immersion bioreactor culture, with the growth ratio reaching 10.9 after 50 days of culture. This was obviously higher than the ebb and flood bioreactor culture. During bioreactor culture, sucrose and electric conductivity (EC) in the culture medium were negatively correlated with PLBs growth; the highest PLBs fresh and dry biomass was obtained 40 days after culture. An inoculation density of 20 g (FW) was optimal for PLBs growth in a 3 l working volume of 5 l bioreactor. Furthermore, MS medium supplemented with 2.0 mg l⁻¹ BA for shooting and 0.5 mg l⁻¹ IBA for rooting was optimal during in vitro culture, the plantlets were successfully established in the potting substrate.     

Plant regeneration of Nerium oleander L. from alginate-encapsulated shoot explants after short-term cold storage

In this study, an efficient protocol for the regeneration of encapsulated explants of oleander (Nerium oleander L.) has been developed. Shoot tips and 1st nodal segments below the shoot tip, from in vitro-derived oleander microshoots, were encapsulated in 2.5% sodium alginate prepared in liquid MS sucrose-free nutrient medium and hardened in 50 mM of calcium chloride producing solid beads, uniform in shape. These artificial seeds, irrespective of their maintenance under light or in darkness, germinated at frequencies of 38.8–42.2%, producing 3.0–3.3 microshoots per bead. In the case of using 100 mM of calcium chloride for hardening, the beads were firm, of uniform globular shape and suitable for handling, exhibiting a germination response of 68.9%. Encapsulated shoot tip explants, following storage at 4°C for 8 weeks, exhibited a higher regeneration response (60.0%) than non-encapsulated similar explants stored under the same conditions (11.1%). Microshoots, excised from cold-stored encapsulated explants after germination, rooted easily in agar-solidified MS medium with 2 μΜ IBA and after their transplantation into a peat-perlite substrate (3:1, v/v), were acclimatised successfully and established in the greenhouse with minimal losses. The present encapsulation procedure could be applied as an alternative method of micropropagation of desirable elite clones of oleander.     

Micropropagation of Cymbidium Sleeping Nymph through protocorm-like bodies production by thin cell layer culture

Transverse thin cell layers (tTCLs) of protocorm-like bodies of two stages of PLBs (30 d and 60 d old) of Cymbidium Sleeping Nymph were used as explants to induce PLBs by using coconut water (CW) as a natural additive. 5% (v/v) CW supplemented to KC medium induced an average of 5 PLBs per responding tTCL of 30 d old PLBs with 83% of responding tTCLs. A low percentage of responding tTCLs were observed in 60 d old PLBs’ tTCLs. Anatomical and confocal microscopic studies traced the origin of PLBs to subepidermal layers of the tTCL. A significantly high percentage of shoot regeneration was obtained from PLBs formed on 1–10% (v/v) CW from tTCLs of 30 d old PLBs in comparison to PLBs induced on control after first subculture on KC medium (without CW). The induced PLBs regenerated into plantlets with velamenous roots and these plantlets were transferred to greenhouse conditions on cocopeat:perlite (9:1) with nearly 100% survival. Post-transfer performance of the plantlets was monitored. The results suggest tTCLs as potential explants (with respect to economy of precious hybrid materials) which can overcome the slow growth of hybrid PLBs and coconut water as a single natural additive for the mass multiplication of commercially important orchids.     

Medium-term conservation of redwood (Sequoia sempervirens (D. Don.) Endl.) in vitro shoot cultures and encapsulated buds

This study evaluated the survival and recovery of non-encapsulated and encapsulated shoots of Sequoia sempervirens after storage at 4 °C in the dark for up to 15 months on four different culture media. Survival and regrowth of encapsulated shoots declined within 3 months, regardless of the storage medium composition. By contrast, no significant decrease in survival and regrowth was noted with non-encapsulated shoots after 12 months of storage on Quoirin and Lepoivre medium supplemented, or not, with 1 mg l⁻¹ benzyladenine. Regrowth dropped to 60–61% after 15 months of storage on the same media. Medium-term conservation of S. sempervirens germplasm is therefore possible using in vitro storage of non-encapsulated shoot cultures.     

Micrografting of almond (Prunus dulcis Mill.) cultivars “Ferragnes” and “Ferraduel”

The success of various in vitro micrografting techniques, establishment of the rootstock, size of the microscion, and the effects of culture medium on the grafted seedling development for almond cultivars “Ferragnes” and “Ferraduel” were studied. In vitro germinated wild almond seedlings developed from seeds were used as rootstocks. Shoot culture initiation was successfully achieved from the above almond cultivars by culturing mature shoot tips from forced nodal buds, about 3–5 mm, on 0.7 mg/L BA and 0.01 mg/L NAA containing a MS medium. The regenerated adventitious shoots from in vitro cultures were maintained and proliferated by sub-culturing on a fresh medium every three to 4 weeks. Regenerated shoot tips, which were micrografted onto in vitro seedlings, resulted in the restoration of shoot proliferation. The results indicated that the most successful method for the grafting of tested almond cultivars was slit micrografting. High levels of micrograft take were achieved with all ranges of scions (4–15 mm) obtained from the regenerated shoot tips. Slow growth and lack of axillary shoot development on the micrografts were noticeable when the micrografts were cultured on hormone-free germination medium. In vitro micrografted plantlets were successfully acclimatized and no problems were encountered with the establishment of micrografted plants in vivo. The developed technique has demonstrated a high potential for application in the micropropagation of almond cvs. “Ferragnes” and “Ferraduel” and thereby, represents a feasible method for the renewal of almond orchards in Turkey and elsewhere in the world.     

Influence of ventilation and sucrose on growth and leaf anatomy of micropropagated potato plantlets

Potato single nodes were cultured in vessels containing MS medium supplemented with 10, 20 and 30 g/l of sucrose. Vessels were closed with a clear polypropylene lid with or without 10 mm microporous polypropylene membrane. Sucrose concentration significantly increased plantlet height, shoot fresh weight and chlorophyll a content. Plantlets grown in ventilated vessels were significantly shorter, had lower shoot fresh weight and higher shoot dry weight than those in non-ventilated vessels. The highest leaf chlorophyll a content (21.83 mg/g fresh weight) was found in plantlets grown in ventilated vessels using MS medium with 20 g/l of sucrose, whereas those grown on medium with 10 g/l of sucrose had the highest chlorophyll b content (24.00 mg/g fresh weight). Total chlorophyll content was significantly higher when plantlets were grown in ventilated vessels containing medium with 10 or 30 g/l sucrose than in non-ventilated vessels. There was no significant difference in total chlorophyll content among plantlets grown in ventilated vessels with different concentrations of sucrose. Stomatal density was significantly lower when plants were grown under ventilated conditions. Leaf replica examination showed that stomata under non-ventilated condition were spherical with wide openings whereas, those in ventilated vessels were elliptical with narrow openings. Plantlets grown in non-ventilated vessels had thinner leaves and failed to build up a distinct defined upper epidermis, palisade parenchyma layer and spongy cells. On the other hand, leaves under ventilated conditions showed comparatively well organized layers with small intercellular space. The vascular system of leaves under the ventilated conditions demonstrated very well developed xylem unlike leaves under non-ventilated conditions. Thus, ventilated vessels with the 20 g/l of sucrose under ambient CO₂ in the growth room could successfully promote photomixotrophic culture and produce healthy plantlets.     

A micropropagation system for cloning of Bambusa tulda Roxb.

The communication describes standardization of an efficient in vitro propagation and hardening procedure for obtaining plantlets from field grown culms of Bambusa tulda. Administration for 10 min of 0.05 and 0.1% mercuric chloride to explants collected in winter and summer seasons, respectively facilitated optimum culture establishment and bud break. 0.1–0.2% mercuric chloride in rainy season enhanced aseptic culture establishment but inhibited bud break due to toxicity to explants. MS liquid medium enriched with 100 μM glutamine, 0.1 μM indole-3-acetic acid and 12 μM 6-benzylaminopurine supported maximum in vitro shoot multiplication rate of two-fold. The proliferated shoots were successfully rooted on MS liquid medium supplemented with 40 μM coumarin resulting in a maximum of 98% rooting. The procedure requires 45 days cycle for the in vitro clonal propagation (15 days for shoot multiplication and 30 days for root induction) and 80 days for acclimatized plantlet production.     

In vitro propagation of Lychnis senno Siebold et Zucc., a rare plant with potential ornamental value

Lychnis senno is a rare and valued ornamental plant. Seed propagation is not efficient because of the low germination rate. To grow commercially L. senno in China, a protocol for in vitro germination and propagation of this species was developed. Various germination rates were obtained by treating seeds with GA₃ during 1–6 months storage period. The highest germination rate reached 19.4% when seeds were treated with 250 mg/l GA₃ and stored for 5 months at 4 °C. Axillary shoot proliferation was induced in the nodal segments of the seedlings on medium containing specific concentrations of BA and NAA [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant 15, 473–497]. Maximum number of shoots was developed on a medium supplemented with 5 mg/l BA and 0.5 mg/l NAA, while the higher shoots were observed on a medium supplemented with 0.5 mg/l BA and 0.05 mg/l NAA. Rooting was induced in 91.7% of the regenerated explants on a half-strength MS medium supplemented with 0.5 mg/l NAA. The plantlets grew well and flowered after transfer to the greenhouse. The chromosome numbers of seedlings and propagated plants were also determined to be 2n = 2x = 24.