Genetic fidelity assessment of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated plants of <Emphasis Type="Italic">Albizia julibrissin</Emphasis> using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2?±?3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3?±?6.3%). Maximum rooting of shoots (73.3?±?5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.     

High frequency in vitro regeneration system for conservation of Coleus forskohlii: a threatened medicinal herb

Present study reports a high frequency regeneration system for in vitro propagation and conservation of an important and threatened medicinal herb Coleus forskohlii (Briq.). Shoot multiplication has been achieved through axillary bud development and direct adventitious shoot formation in nodal explants on Murashige and Skoog (MS) medium containing 6-benzyladenine (BA) (5 μM). Further shoot multiplication was recorded up to third subculture on MS medium containing BA (5 μM) in combination with 1-naphthleneacetic acid (NAA) (0.1 μM). Excised microshoots on transfer to root induction medium consisting of half-strength MS medium (1/2 MS) alone as well as in combination with various auxins, resulted in varied rooting pattern in terms of number, length, and type of roots. Rooted microshoots were acclimatized successfully in earthen pots containing garden manure, garden soil, and sand (1:2:1) as potting mix with survival rate of 70 %. Acclimatized plantlets were studied for the amount of chlorophyll and carotenoid content as well as the net photosynthetic rate (P_N) during subsequent weeks of transfer to ex vitro condition. Histological studies revealed the direct origin and development of shoot buds from basal swollen cut end of nodal explants.     

In vitro propagation of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L. by somatic embryogenesis in cell suspension culture

An effective protocol was developed for in vitro propagation of Psoralea corylifolia via somatic embryogenesis in cell suspension culture. Embryogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 6 μM naphthaleneacetic acid (NAA) and 30 μM glutamine from transverse TCLs from 10-day-old hypocotyl explants with a 96.4% frequency. Embryogenic callus produced a higher number of somatic embryos (123.7 ± 1.24 per gram fresh weight callus) on MS medium containing 30 g l^?1 sucrose, 1 μM NAA, 4 μM benzyladenine (BA), 15 μM glutamine and 2 μM abscisic acid (ABA) after 4 weeks of culture. Somatic embryos successfully germinated (97.6%) on ½ MS medium containing 20 g l^?1 sucrose, 8 g l^?1 agar and supplemented with 2 μM BA, 1 μM ABA and 2 μM gibberellic acid (GA₃) within 2 weeks of culture. Somatic embryos developed into normal plants, which hardened with 100% efficiency in soil in a growth chamber. Plants were successfully transferred to greenhouse and subsequently established in the field. Plant survival percentage in the field differed with seasonal variations. Average psoralen content of 12.9 μg g^?1 DW was measured in different stages of somatic embryo development by high-performance liquid chromatography (HPLC). This protocol will be helpful for efficient propagation of elite clones on a mass scale, conservation efforts of this species and for secondary metabolites production studies.     

Factors affecting plantlet regeneration from in vitro cultured immature embryos and cotyledons of Prunus mume “Xue mei”

Using immature embryos and cotyledons as explants, a successful immature embryo culture and efficient plant direct regeneration via organogenesis from cotyledons, which showed different patterns, was established for the “Xuemei” cultivar of Prunus mume. For immature embryo culture, high frequency plantlet forming (89.5%) from embryo axis was obtained on half-strength Murashige and Skoog (½MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). At the same time, shoots direct differentiation from cotyledons with the embryo axis development was also observed on ½MS medium containing 2.2 μM BA together with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when embryo axes were removed from cotyledons and cultured on ½MS medium supplement with 13.2 μM BA, 2.7 μM NAA (72.9%) or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA (84.2%), respectively. Regenerated shoots were successfully rooted on ½MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of embryo axes, BA and TDZ, on cotyledons’ regeneration were investigated in detail. The rooted plantlets were transferred to soil successfully with normal morphology.     

Plant regeneration from mesophyll protoplasts of a medicinal plant, Phellodendron amurense rupr.

Summary A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×10⁵ protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×10⁵−6×10⁵ protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.     

Somatic embryogenesis in banana (Musa acuminata AAA cv. Grand Naine): effect of explant and culture conditions

An improved, rapid, reproducible, and simple protocol has been developed for somatic embryogenesis in banana cv. ‘Grand Naine’ using explants derived from actively growing multiple shoot cultures. Many restrictive factors remain in banana embryogenesis such as long duration, unpredictability, and a high degree of genotype dependence. In the present study, we used split shoot tips from 4-wk-old cultures as explants. Somatic embryos were induced in 15 d directly in Murashige and Skoog (MS) medium supplemented with different combinations of 0–8.28 μM picloram and 0.22–4.44 μM 6-benzylaminopurine (BA) without callus formation. Maximum embryo induction (100%) occurred when 4.14 μM picloram and 0.22 μM BA were used. Conversion of somatic embryos into plantlets occurred sporadically (2–3%) in MS medium containing α-naphthalene acetic acid (NAA; 0.53–2.68 μM) together with BA (2.22–44.39 μM), or thidiazuron (4.54 μM) plus glutamine (200 mg/L). This protocol is far superior to those already reported for fast and high frequency induction of somatic embryo. In liquid agitated culture, individual embryos separated easily and produced a large number of secondary embryos within 10 d which, upon transfer to filter paper overlaid on MS liquid medium supplemented with 4.44 μM BA, resulted in conversion (3%) into plantlets.     

Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Manchurian ash (Fraxinus mandshurica Rupr.)

Using mature cotyledonary explants of Fraxinus mandshurica, an efficient plant regeneration system was developed via somatic embryogenesis. More than 67 % of mature cotyledons of zygotic embryos yielded 23–159 somatic embryos (SEs) per explant when incubated on medium consisting of half-strength Murashige and Skoog (MS) salts and vitamins (MS1/2) supplemented with 8.88 μM 6-benzyladenine (BA), 26.84 μM naphthaleneacetic acid (NAA), 75 g L^?1 sucrose, and 400 mg L^?1 casein hydrolysate (CH). Approximately, 82 % of induced SEs were observed on browning cotyledonary explants. Histological studies of cotyledon explants at various stages of somatic embryogenesis revealed that the SEs originated from single epidermal cells and developed to the globular, heart, torpedo, and cotyledonary stage embryos. Secondary somatic embryos (SSEs) formed on the surface of radicle tips of the SEs. Addition of low concentrations of NAA and 200–400 mg L^?1 CH to MS1/2 medium increased SSE induction. Cotyledonary SSEs were cultured on MS1/2 medium with 10 mM abscisic acid in the presence of light to promote maturation, and >92 % of mature SSEs were able to germinate with normal shoots. After 8 weeks in culture in the presence of light on medium with one-third of the MS macroelements as well as 0.06 μM NAA, >94 % of the germinated SSEs converted into plantlets. Plantlets acclimatized successfully to ex vitro conditions and developed normal phenotypes under field conditions.     

Effects of culture conditions on multiple shoot induction from inflorescence and RAPD analysis of cloned plants in Limonium sinensis (Girard) Kuntze, var. Golden Diamond

The optimum physical and chemical microenvironment for micropropagation of Limonium sinensis (Girard) Kuntze, var. Golden Diamond was established from immature inflorescence segments as explant. The highest frequency (62 %) of axillary shoot induction was obtained on MS medium (Murashige and Skoog Physiol Plant 15:473–497, 1962) supplemented with 8.88 μM BA, 1.34 μM of NAA and two growth additives cysteine (142.33 μM), and glutamine (684.22 μM). In the subsequent culture maximum average number of shoots (11.13?±?0.34) were obtained from micro-shoot explant on MS medium supplemented with the same additives and 2.22 μM BA. During subcultures the problem of vitrification was mitigated through increasing agar concentration from 0.8 % to 1.0 % and providing better ventilation. The in vitro developed shoots were rooted on MS medium supplemented with 2.46 μM IBA and 0.88 μM BA. Rooted plants were acclimatized successfully in the greenhouse with 80 % survival rate. RAPD analysis using 15 random decamer primers generated monomorphic banding pattern in micropropagated plants and similar to those of mother plant revealing the genetic integrity of regenerants.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration and enhanced synthesis of plumbagin in root callus of <Emphasis Type="Italic">Plumbago zeylanica</Emphasis> L.—an important medicinal herb

Plumbago zeylanica L., an important medicinal herb, possesses plumbagin, a valuable secondary metabolite. Roots of this plant, collected from four locations in Himachal Pradesh, India, were screened for plumbagin content with high-performance liquid chromatography. The chemotype collected from Hamirpur yielded the highest content (26.47?±?0.63 mg g^?1 dry weight). Callus cultures were established from nodal explants of this chemotype on Murashige and Skoog (MS) medium augmented with α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid, (2,4-D), 6-benzyladenine (BA), isopentenyl adenine (2iP), or thidiazuron, (TDZ). After 45 d, 98% of the cultures induced bright-green, compact callus on MS?+?5 μM TDZ. Upon subculturing, this callus differentiated an average of 4.08?±?1.16 shoots in 62.5% of the cultures. After elongation on basal MS medium, excised shoots were transferred to indole-3-acetic acid, NAA, or IBA supplemented MS medium. A maximum of 4.3?±?1.36 roots with an average length of 15.31?±?2.76 cm were recorded on 5 μM IBA. Rooted plantlets were successfully acclimatized in a greenhouse, and their genetic fidelity was evaluated using inter simple sequence repeats and start codon targeted molecular markers, which revealed 97% similarity. A significant increase in plumbagin content (6.5- and 3.4-fold) was achieved in root callus employing 100 mg L^?1 yeast extract (YE) and 25 μM salicylic acid (SA), respectively. This is the first report of large-scale propagation of P. zeylanica and an increase of plumbagin through in vitro root callus.     

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.     

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum,an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

Plant regeneration from protoplasts in Indian local Coriandrum sativum L.: scanning electron microscopy and histological evidences for somatic embryogenesis

Coriandrum sativum L. is an annual herb belonging to the family Umbelliferae. It is used as a spice plant in Indian subcontinent and it has several medicinal applications as well. In this present article, an efficient plant regeneration protocol from protoplasts via somatic embryogenesis was established and is reported. This is the first ever protoplast isolation study in Indian local coriander in which plant regeneration was achieved. Hypocotyl-derived embryogenic callus was used as a source of protoplast. The embryogenic callus suspension was prepared by transferring tissues onto rotary-agitated liquid Murashige and Skoog, added with 1.0 mg l^?1 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l^?1 KIN (6-furfurylaminopurine). The suspension was digested with enzymatic solutions and a combination of cellulase (2.0 %), pectinase (1.0 %), macerozyme (0.02 %) and driselase (0.50 %) induced maximum yield of protoplasts (34.25 × 10⁵). In 1.0 mg l^?1 2,4-D + 1.0 mg l^?1 KIN containing medium, protoplasts divided well and formed maximum number of microcolonies (14.30/test tube). The protoplast callus (PC) biomass grew well in solid medium. The protoplast embryogenic callus was rich in protein, proline and sugar compared to non-embryogenic PC. The protoplast originated callus later differentiated into somatic embryos. The somatic embryo morphology, scanning electron microscopy and histology of embryo origin and development were investigated and discussed in details in this present communication. In 1.0 mg l^?1 2,4-D + 0.5 mg l^?1 BA (6-Benzyladenine), maximum number of embryos were formed on microcallus (26.6/callus mass). The embryo matured and germinated into plantlets at a low to moderate rate, highest (31.3 %) embryo germination was observed in 1.0 mg l^?1 BA + 0.5 mg l^?1 α-Naphthalene acetic acid added medium. The entire process of regeneration took about 4–5 months’ time for recovering plantlets from protoplasts.     

Isolation,culture and plantlet regeneration from cotyledon and mesophyll protoplasts of two pickling cucumber (Cucumis sativus L.) genotypes

Optimal protoplast yields from cotyledons (2.0×10⁶ protoplasts/ 0.5 g tissue) and from true leaves (5.0×10⁶ protoplasts/g tissue) of two Cucumis sativus genotypes were obtained following a 16 h digestion with, respectively, 1.25% pectinase+0.5% Cellulysin and 0.5 % pectinase+ 1.0% Cellulysin. Enzyme solutions were prepared in modified MS medium containing half-strength major salts, full complement of minor salts and vitamins, 2% sucrose and 0.25 M mannitol. A plating density of 3.5–4.0× 10⁴ protoplasts/ml or higher was required for sustained division, with first division occurring in 6–7 days, second-third division in 8–9 days, and minicalli formation by day 13. Embedding in 0.4% agarose provided the highest plating efficiency (proportion that formed minicalli) of mesophyll protoplasts, which was 28.3% for genotype 3672 and 15% for genotype 3676. By comparison, liquid culture and droplet culture gave lower plating efficiencies (10–19%). Cotyledon and mesophyll protoplasts of one genotype formed minicalli on MS medium containing 2,4-D/BA at 1.0/2.5 M and 5.0/5.0 M, respectively, within 21 days, while mesophyll protoplasts of the second genotype formed minicalli on MS medium containing NAA/BA at 5.0/5.0 M within 12 days. Shoot buds or somatic embryos were obtained upon subculture of calli to MS medium containing lower concentrations (0.05–0.01 M) of 2,4-D/BA or NAA/BA and a few plantlets, ca.18, were recovered on hormone-free medium.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid     

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.     

In vitro propagation, proscillaridin A production and antibacterial activity in Drimia robusta

Drimia robusta is a threatened traditional medicinal plant extensively used in South Africa. Rapid in vitro mass propagation of the species was developed for commercial cultivation from leaf explants using various concentrations and combinations of plant growth regulators and organic elicitors. The highest number of regenerated shoots per explant (14.6 ± 0.54) was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 2.27 μM thidiazuron (TDZ), 2.22 μM benzyladenine (BA) and 20 μM glutamine. Adventitious shoots were rooted and the plantlets were successfully acclimatized (100 %) in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Proscillaridin A (PsA) content and the antibacterial activity of in vitro and ex vitro regenerated plants were evaluated in different tissues in comparison to naturally-grown plants. The highest content of PsA (19.68 μg mg^?1 DW) was recorded in roots of ex vitro plants which were grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 100 mg l^?1 casein hydrolysate. In vitro regenerated plants grown on MS medium containing 2.27 μM TDZ, 2.22 μM BA and 50.8 μM MBZ gave high antibacterial activity (MIC of 0.156 mg ml^?1) against both Gram-positive and Gram-negative bacteria. Using this protocol the regenerated plants can be used in traditional medicine as an alternative to naturally collected plants.     

Callus induction and plant regeneration from anthers of Dendrocalamus latiflorus Munro

Bamboo varieties are very difficult to improve by traditional breeding methods. Here, we established an efficient plant-regeneration system for Dendrocalamus latiflorus (tropical giant bamboo) by anther culture. Culture conditions, especially the plant growth regulators required for callus induction and shoot differentiation, were optimized by orthogonal design. M8 medium supplemented with 5.37 μΜ α-naphthaleneacetic acid (NAA), 1.33 μM?N ⁶ -benzyladenine (BA), 110.17 μM phenylacetic acid (PAA), and a pretreatment time of 3 d produced the highest rate (5.08?±?0.61%) of callus induction. The maximum shoot differentiation rate reached 28.3?±?4.29% in M8 medium supplemented with 2.32 μM kinetin (KT), 8.89 μM BA, 1.08 μM NAA, and 110.17 μM PAA. The results of the ploidy level test showed that most of the regenerated plants were dodecaploid (96/100), a few were hexaploid (3/100), and one was triploid (1/100). The average chlorophyll content of dodecaploid lines was significantly higher than that of hexaploid lines. The present study provides an innovative method for bamboo ploidy breeding and a useful method for genetic improvement.     

Efficient regeneration of plantlets from callus and mesophyll derived protoplasts of <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.

A protocol for plant regeneration from mesophyll and callus protoplasts of Robinia pseudoacacia L. was developed. For leaves from in vitro raised shoots, an enzyme combination of 2.0% cellulose and 0.3% macerozyme for a digestion period of 20 h resulted in the best yield of protoplasts (9.45 × 10⁵ protoplast/g fresh weight). Mesophyll-derived protoplasts started cell wall regeneration within 24 h of being embedded in Nagata and Takebe (NT) medium supplemented with 5 μM NAA and 1 μM BAP followed by the first cell division on day three of culture and micro-colony (32 cells) formation within day 7–10 in the same medium. However, using callus as the starting material, a combination of 2.0% cellulose and 1.0% macerozyme for a digestion period of 24 h gave the highest protoplast yield (3.2 × 10⁵ protoplast/g fresh weight). Cell wall regeneration in callus-derived protoplasts started within 24 h followed by the first cell division on the day three (96 h) and the appearance of microcolonies of more than 32 cells by the end of first week (144 h) of culture on solid WPM medium supplemented with 5 μM NAA and 1 μM BAP. Microcalli were visible to the naked eye after 45 days on solid WPM medium. Proliferation of macro-calli was successfully accomplished on solid Murashige and Skoog (MS) medium with 5 μM NAA and 5 μM BAP. Both mesophyll and callus protoplast-derived calli produced shoots on MS medium with 0.5 μM NAA and 1 μM BAP within 25–30 days and multiplied on MS medium with 1.25 μM BAP. Excised microshoots were dipped in 1–2 ml of 2.0 μM IBA for 24 h under dark aseptic conditions and transferred to double sterilized sand for rooting. The flasks containing sand were inoculated with Rhizobium for in vitro nodulation. Forty-five plants transferred to pots in the glasshouse established well.     

High-frequency plantlet regeneration and multiple shoot induction from cultured immature seeds of Rhynchostylis retusa Blume., an exquisite orchid

Seeds of an exquisite orchid, Rhynchostylis retusa, germinated in vitro on ½ Murashige and Skoog (MS) medium supplemented with different concentrations of coconut milk (CM). Of the different concentrations of CM employed for seed germination, 15% gave optimum response. On this medium a maximum of 93% cultures produced seedlings 90 days after inoculation. Individual seedlings with a length of about 0.5 cm were subcultured on MS medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthalene acetic acid (NAA), with or without activated charcoal (AC), for further growth. Seedling growth was maximum on MS medium supplemented with 6 μM BA, 0.2 μM NAA, and 1 g L^?1 AC. Here a maximum seedling length of 2.3 cm was observed after 1 month of culture. The seedlings were subcultured on MS medium supplemented with kinetin (Kn) or thidiazuron (TDZ), in the presence or absence of AC, for multiple shoot induction. A maximum multiple shoot number of 8.2 was observed on MS medium supplemented with 2 μM TDZ in the presence of AC. The shoots were rooted on ½ MS medium supplemented with 2 μM indole-3-butyric acid (IBA) and successfully transplanted to soil. Of the 45 plantlets transferred to soil 40 survived. The reproducible protocol standardized here will enable rapid propagation and conservation of this precious orchid.     

Asymbiotic seed germination and in vitro seedling development of Cymbidium aloifolium (L.) Sw.: a multipurpose orchid

Cymbidium aloifolium is a multipurpose economically important epiphytic orchid grows on tree trunk in the primary forests. Its population in natural habitat is downsized due to different anthropogenic activities. A successful attempt was made for asymbiotic immature embryo culture and in vitro mass scale production of plantlets. For successful culture initiation seed pods of various developmental ages, various nutrient media, sucrose concentrations, different quality and quantity of plant growth regulators were surveyed. Immature embryos of 9 months after pollination was successfully germinated on MS medium containing sucrose (2%) (w/v) and α-naphthalene acetic acid (NAA) and benzyl adenine (BA) (3 and 6 μM respectively in combination) within 45 days of culture where 90% germination was recorded. The germinated seeds formed PLBs on the optimum germination medium within two passages. The protocorm like bodies (PLBs) differentiated into rooted plantlets within 3 weeks on regeneration medium containing sucrose (3%), casein-hydrolysate (0.1 gl^?1) and BA 3 μM. Amongst the three media studied, optimum regeneration was registered on MS medium where as many as 12 shoot buds developed per explants per subculture of 4 weeks duration. The well rooted plantlets of 6–7 cm long with 3–4 roots were hardened in vitro 3–4 weeks before they were transferred to potting mix. The potted plants were exposed to full sunlight periodically and watered at regular interval. About 70–80% transplants survived after 2 months of potting.