Rapid regeneration of plants of Dendrobium lituiflorum Lindl. (Orchidaceae) by using banana extract

Effects of banana extract (BE) and 6-benzylaminopurine (BAP) were evaluated on asymbiotic seed germination and an early differentiation of protocorms and plant regeneration of Dendrobium lituiflorum Lindl. High percentage germination was achieved by culturing seeds on modified Knudson C medium supplemented with 10% (v/v) BE. Rapid regeneration was observed within 60 days of culture on 10% (v/v) BE supplemented KC medium where maximum percentage propagules showed development of leaves and root formation. Propagules on BAP supplemented KC medium showed no further development beyond one leaf stage. In another experiment, culture of shoots on 12.5% (v/v) BE supplemented KC medium led to multiplication, shoot elongation as well as vigorous rooting. Shoots cultured on 10 μM BAP supplemented MS medium showed maximum multiplication but these were stunted. Plants with well expanded deep green leaves and elongated roots from BE media were first hardened in vitro followed by ex vitro hardening on cocopeat:perlite (9:1) in the greenhouse conditions and exhibited 90% survival. The study emphasizes the role of BE as a natural additive at different stages of development from seed germination to plant regeneration.     

Effects of light,sucrose, and cytokinins on somatic embryogenesis in Lilium ledebourii (Baker) Bioss. via transverse thin cell-layer cultures of bulblet microscales

Summary

This is the first report describing the culture conditions necessary to induce somatic embryogenesis and plantlet regeneration from transverse thin cell-layers (tTCL) of the rare and endangered bulb species, Lilium ledebourii (Baker) Boiss. (Liliaceae). The tTCLs were transferred onto 1.0 Murashige and Skoog medium (MS) containing various sucrose concentrations [3.0, 4.5, or 6.0% (w/v)] and different combinations of two cytokinins [6-benzylaminopurine (BA) or thidiazuron (TDZ)] with 1.0 µM -naphthaleneacetic acid (NAA) in the dark, or exposed to light (40 µmol m^–2 s^–1). The aims of this work were to provide an improved propagation method torescue L. ledebourii, and to determine the effects of sucrose concentration, light, and different cytokinins on somatic embryogenesis. Embryogenic callus cultures were obtained only when the tTCLs were transferred onto 1.0 MS medium containing 1.0 µM NAA, various levels of BA (0.4, 1.1, or 2.2 µM), and sucrose [3.0, 4.5, or 6.0% (w/v)] after 3 months culture in the light or in darkness. Combinations of various concentrations of TDZ and NAA did not generate embryogenic calli. The highest rate of growth of embryogenic calli was achieved on 1.0 MS medium supplemented with 1.0µM NAA, 1.1 µM BA, and 4.5% (w/v) sucrose, in the light. Embryo-like structures were grown into plantlets after transfer onto 1.0 MS medium without any plant growth regulators and incubated in the light. Regenerated plants were successfully acclimatised to ex vitro conditions, with a survival rate of 90%.     

Somatic embryogenesis and plantlet regeneration from encapsulated embryos of Selinum tenuifolium

Summary

This paper reports, for the first time, somatic embryogenesis and synthetic seed production in Selinum tenuifolium Wall. Mature leaf explants inoculated in Murishige and Skoog (MS) medium supplemented with 3 µM 2,4-dichlorophenoxyacetic acid (2,4-D), containing 3% (w/v) sucrose and 0.7% (w/v) agar, induced 67% callus. Maximum production of globular structures, their differentiation into embryos and germination, occurred with a combination of 2 µM benzyladenine (BA) and 2 µM indole-3-butyric acid (IBA). To protect somatic embryos and produce synthetic seeds, gel capsules were standardised using a combination of sodium alginate and calcium nitrate concentrations. Gel capsules were most effective when formed with a combination of 3% (w/v) sodium alginate and 100 mM calcium nitrate for 30 min. The addition of MS medium to alginate capsules with 3% (w/v) sodium alginate, 3% (w/v) sucrose, 2 µM BA and 2 µM IBA significantly improved their germination rate to 77.8%, as well as their resulting shoot length (5.6 cm) and root length (7.2 cm), compared to controls (57.8%). Most plantlets (66%) survived under nursery condition. Storage at 4°C for different periods (10 d or 20 d) significantly (P < 0.05) reduced the percentage survival and germination of somatic embryos and artificial seeds compared to controls or 5 d storage.     

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.     

Evaluation of the efficiency of somatic embryogenesis in guava (Psidium guajava L.)

Summary

An embryogenic protocol for plant regeneration of guava (Psidium guajava L.) was established using 10-week post-anthesis, zygotic embryo explants. Somatic embryogenesis was induced on Murashige and Skoog medium (MS) containing 3% (w/v) sucrose, 0.8% (w/v) agar and various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) by continuous treatment of the zygotic embryo explants. Somatic embryos appeared as globular structures at the end of the third week from culture initiation, and heart-shaped, cotyledonary-stage, and torpedo-stage embryos appeared within the next few weeks. The development of somatic embryos was asynchronous and showed five-to-seven discernible stages. Depending upon the response of the somatic embryos during their maturation, germination, acclimatisation, and encapsulation, they were grouped into one of three categories. The preferred type of somatic embryos (≥ 1.5 mm) were called the “elongated torpedo” (ET) category. The slightly less-preferred type of stomatic embryos (from 1.0 – 1.5 mm) were termed the “short torpedo” (ST) category. The least preferred types of somatic embryos, at the cotyledonary, heart-shaped, and/or globular stages of development (< 1.0 mm), were grouped into a third category designated “CHG”. The suitability and efficacy of various growth regulators and other treatments were assessed based on six different embryogenic parameters: (i) the frequency of embryogenesis; (ii) the intensity of embryogenesis, defined as the average number of somatic embryos produced per culture (“ANEPC”); (iii) the frequency of ET somatic embryos; (iv) the frequency of ST somatic embryos; (v) the frequency of CHG somatic embryos; and (vi) the overall efficiency of embryogenesis, defined as the potential of a treament to produce somatic embryos at the ET or ST stages, or at both stages of development, that could be converted into plantlets. In the present report, we found that 0.01 mg l^–1 2,4-D gave the maximum frequency and intensity of embryogenesis. But the highest frequencies of ET and ST somatic embryos were produced on MS medium containing 3% (w/v) sucrose and 0.001 mg l^–1 2,4-D, while CHG embryos were produced at the highest frequency on the same medium, but containing 0.5 mg l^–1 2,4-D. It was difficult to calculate the most effective concentration of 2,4-D for somatic embryogenesis based on parameters (i) – (v) above. Hence, quantitative estimations of the efficiency of embryogenesis (sixth parameter) were imperative in order to analyse the potential of the different treatments. The highest efficiency of somatic embryogenesis was achieved by continuous treatment of 10-week post-anthesis, zygotic embryo explants with 0.01 mg l^–1 2,4-D on full-strength MS agar medium containing 3% (w/v) sucrose. These somatic embryos matured normally on the same medium, and germinated well both on half-strength solid and in half-strength liquid MS medium containing 3% (w/v) sucrose. They grew in full-strength liquid MS medium with 3% (w/v) sucrose and showed maximum survival upon transfer to soil and hardening. Evaluations of the efficiency of somatic embryogenesis in guava, based on the six parameters defined above, have also helped us to understand and evaluate processes for high efficiency micropropagation in other species.     

High-frequency embryogenesis,regeneration of broccoli (Brassica oleracea var. italica) and analysis of genetic stability by RAPD

High-frequency somatic embryogenesis and shoot regeneration of broccoli (Brassica oleracea var. italica) were achieved. Cotyledon and hypocotyl explants from four varieties of broccoli were cultured on MS and modified MS media (mMS, supplemented with PG-96 organic components) with different combinations of growth regulator. The effects of genotypes, different explants, growth regulator combinations, organic components and AgNO₃ on induction of calli and shoots were evaluated. The optimal media for inducting calli/shoots and roots were mMS medium containing 3% (w/v) sucrose and 0.8% (w/v) agar supplemented with NAA at 0.5 mg l⁻¹, 6-BA at 3.0 mg l⁻¹, AgNO₃ at 4.0 mg l⁻¹ and MS medium containing 3% sucrose and 0.8% (w/v) agar supplemented with NAA at 0.2 mg l⁻¹, respectively. The callus induction percentages were over 90% in all four varieties; shoot induction percentage was 92.5% and the average number of shoot per explant was 4.1 from cotyledon explant in variety Bishan. In this study, we established high-efficient embryogenesis and shoot regeneration system of broccoli and analyzed genetic stability of regenerants at DNA level using RAPD molecular marker. Out of 62 arbitrary primers screened using PCR amplification, 79 polymorphic bands were amplified from 20 primers. The results demonstrated the genetic stability of regenerants from the same variety.     

车轮梅茎段高效再生体系的建立

 以车轮梅 (Rhaphiolepis indica L.)茎段为外植体。探讨基本培养基种类、植物生长调节剂组合、蔗糖浓度和AgNO3等因素对茎段器官发生和植株再生的影响。结果表明：MS+6-BA 2.0 mg·L^-1 +NAA 0.5 mg·L^-1 + AgNO3 1.0 mg·L^-1+蔗糖40 g·L^-1培养基最适合不定芽的分化和增殖,不定芽分化率达90%以上,平均每外植体分化不定芽数达5.38个。不定芽可在1/2MS培养基中有效伸长,适宜生根培养基为1/2MS+NAA 1.0 mg·L^-1,生根率达到100%。     

Black Currant Leaf Spot: II. Laboratory Tests of Fungicides for the Prevention of Sporing of Pseudopeziza Ribis on Overwintered Leaves

Summary

We have developed a two-step procedure for rooting of tea microshoots in vitro. The effectiveness of different auxin treatments for root formation was found to differ. Among the auxins tested, 25 μM -naphthalene acetic acid (NAA) gave the best results, with 100% rooting, compared to 25 μM indole-3-butyric acid (IBA) or 25 μM indole-3-acetic acid (IAA), which induced 17% and 58% rooting, respectively. Incubation of tea microshoots on 0.33 Murashige and Skoog (MS) medium, supplemented with 25.0 μM NAA or 175.0 μM IBA for 10 d, followed by transfer to auxin-free 0.33 MS medium resulted in 100% rooting, whereas 50.0 μM IAA induced 91.7% rooting. Besides the different auxin treatments, the strength of the MS medium, the duration of incubation of microshoots in auxin-containing medium, the sucrose concentration, the gelling agent, the pH of the medium, the incubation temperature, the light intensity, and the quality of the shoots also played a significant role during in vitro rooting of micropropagated tea shoots. Among the combinations tested, the most effective results were obtained when green microshoots were incubated on 0.33 MS medium supplemented with 25.0 μM NAA, 50.0 mM sucrose, pH 5.5, gelled with 0.2% (w/v) Phytagel^TM for 10 d at 25° – 30°C at a light intensity of 40 μmol m^–2 s^–1, followed by transfer of shoots to auxin-free 0.3 MS medium. This resulted in 100% rooting and, on average, 11 long roots were formed per shoot. Anatomical changes during adventitious rooting of micropropagated tea shoots in vitro were also studied to understand the process of rooting.     

In vitro asymbiotic seed germination and seedling growth of Ansellia africana Lindl.

The effects of sodium hypochlorite treatment and growth medium on in vitro seed germination and seedling growth of the leopard orchid (Ansellia africana Lindl.) were investigated. Forty minutes treatment of seeds with 1.75% sodium hypochlorite stimulated germination (70.6%) and seedling development when measured at Stage 5 (emergence of first leaf) on P668 medium after 8 weeks of culture. The growth medium played a significant role in determining the germination response of A. africana seeds. Dark pretreatment of seed cultures significantly enhanced the germination percentage and the growth of rhizoids on the protocorms. Leaf growth in terms of length was substantially enhanced on P668 medium. It was significantly reduced in modified Knudson C medium after 16 weeks of culture. Seedlings developed on P668 medium showed a significantly better growth performance in relation to leaf length, leaf number, root length, fresh and dry weights per plant in vermiculite after 12 weeks of ex vitro growth in a mist house with 90% relative humidity. Further studies developing a suitable method for in vitro symbiotic germination could assist in reintroduction of this threatened orchid species into the wild.     

Rapid in vitro germination of zygotic embryos of fluted pumpkin (Telfairia occidentalis Hook. f.)

Summary

Fluted pumpkin, Telfairia occidentalis, is becoming an important regional vegetable for its food and medicinal uses. The recalcitrant nature of its seed makes conservation difficult and in vitro techniques may be a viable option for conservation. A pilot study was conducted on the effects of different concentrations of a commercial bleach [3.85% (w/v) sodium hypochlorite] for surface sterilisation of T. occidentalis seed. The optimum concentration [25% (v/v)] was then used as a basis to investigate the responses of mature embryonic axes of T. occidentalis to different concentrations of kinetin (Kin; 0, 1.0, or 2.0 mg l^–1) and 1-naphthaleneacetic acid (NAA; 0, 0.5, or 1.0 mg l^–1) combined in a factorial design. The results of the first experiments indicated that commercial bleach at 25% (v/v) resulted in the lowest contamination of explants (10%), with no evident injury to the embryonic axes. The results revealed that root emergence started 3 d after initiation (DAI) only on Murashige and Skoog medium (MS) with no added plant growth regulator (PGR), and that, by 12 DAI, all media supported the rooting of explants. The highest rooting percentage (69%) was observed at 15 DAI on MS medium with 0.5 mg l^–1 NAA, without Kin. However, shoot emergence started at 9 DAI on PGR-free MS medium, on MS with 0.5 mg l^–1 NAA, or on MS plus 1.0 mg l^–1 Kin. The highest shooting percentage (91%) of explants was observed with 0.5 mg l^–1 NAA at 21 DAI. Considering all other growth parameters, MS medium supplemented with 0.5 mg l^–1 NAA was found to be best for the germination of embryonic axes of T. occidentalis.     

Rapid micropropagation of Psoralea corylifolia L. using nodal explants cultured in organic additive-supplemented medium

Summary

The influence of different growth regulators and additives on shoot multiplication from nodal explants of Psoralea corylifolia was investigated. Prolific shoot multiplication was achieved within 4 weeks of culture on Murashige and Skoog (MS) medium supplemented with 5 μM benzyladenine (BA), 5 μM ascorbic acid (AA), 100 mg l^–1 casein hydrolysate (CH) and 5% (v/v) coconut water (CW). Shoots elongated on half-strength MS basal medium devoid of inositol, but containing 5 μM 2-isopentenyladenine (2iP), 10 g l^–1 sucrose and 8 g l^–1 agar. Elongated shoots rooted on half-strength MS basal medium supplemented with 3 μM indole-3-butyric acid (IBA), 10 g l^–1 sucrose and 7 g l^–1 agar within 5 d of culture. The in vitro-raised plants were established successfully in 2:1:1 (v/v/v) garden soil:farmyard soil:sand, and maintained in a growth chamber with 100% survival. Acclimatised plants were transferred to a glasshouse and established successfully in the field. Flowers and fruits appeared after 4 months and resembled those on source plants. This system could be used for rapid commercial propagation of P. corylifolia for conservation strategies and to produce phytomedicines.     

Clonal propagation of Poncirus trifoliate through culture of shoot primordia

Summary

In Poncirus trifoliate, a highly efficient clonal propagation system for the culture of shoot primordia was devised. Shoot primordia were induced at the base of hypocotyl tissue cultured on MS medium supplemented with 44.4 µM BA, 3% sucrose and 0.8% agar. In MS liquid medium (44.4 µM BA, 3% sucrose) on a rotary shaker at two revolutions per minute, shoot primordia of Poncirus grew in size and number. Plant regeneration occurred on MS solid medium. Frequency of regeneration was highest on MS basal medium containing 3% sucrose and 0.8% agar. About 75 shoot buds regenerated from one shoot primordium. Histological observations showed that shoot buds arose from cells in the hypodermal layers of the shoot primordium. The shoot bud developed a vascular system, which became connected to the shoot primordium tissue. Regenerated shoots rooted on 1/2 MS basal medium or 1/2 MS medium supplemented with 0.5 or 5.0 µM IBA. These rooted shoots were acclimatized easily under intermittent mist.     

A combination of gum arabic and chitosan can control anthracnose caused by Colletotrichum musae and enhance the shelf-life of banana fruit

Summary

Anthracnose, caused by Colletotrichum musae, is the major disease affecting the quality of banana fruit during storage. To control banana anthracnose, the combined effects of coatings with gum arabic [GA; at 5, 10, 15 or 20% (w/v)] and 0.75% (w/v) chitosan (CH) were investigated and compared to untreated controls. In vitro results showed significant (P < 0.05) inhibition of mycelial growth and conidial germination of C. musae in all combined treatments compared to the untreated controls after 7 d of incubation at room temperature (25°C). However, potato dextrose agar (PDA) plates amended with 10% (w/v) GA gave the most promising results among all test treatments in suppressing mycelial growth (86%) and inhibiting conidial germination (80%), while no effective inhibition of conidial germination was observed in the controls. In vivo results confirmed that 10% (w/v) GA was the optimum concentration to control fruit decay (70%), while showing efficacy on the reduction of growth of C. musae on artificially inoculated banana fruit. The combined coatings of GA + CH also significantly delayed ripening in terms of weight loss, fruit firmness, soluble solids content, and titratable acidity. These results support the possibility of using 10% (w/v) GA combined with 0.75% (w/v) CH as an alternative strategy to control post-harvest anthracnose disease in banana fruit.     

Silver nitrate and adenine sulphate induced high regeneration frequency in the recalcitrant plant Cosmos bipinnatus using cotyledon explants

Plant regeneration ability was studied in the medicinal-ornamental plant, Cosmos bipinnatus ‘Sonata white’, which is a dicotyledonous recalcitrant plant to shoot induction. Cotyledons were used as sources of explants to investigate plant regeneration. High frequency of direct shoot induction was obtained when BA (5 mg/l) and AgNO₃ (5 mg/l) were used in combination with 20 mg/l adenine sulphate (73.8%) in Murashige and Skoog (MS) medium. The highest shoot number per explant (5.7) was induced on MS medium supplemented with 5 mg/l BA, 5 mg/l AgNO₃, and 40 mg/l adenine. Eight week-old shoots were transferred to root induction media containing MS and half-strength MS medium with different concentration of IBA. The highest rate of root induction (70.8%) was obtained on half-strength MS medium with 1.5 mg/l IBA within four weeks. The plantlets were transferred to pot and kept in the greenhouse condition. Seventy percent of the plantlets successfully acclimatised.

Abbreviations: BA, 6-benzylaminopurine; IBA, Indole-3-butyric acid; MS, Murashige and Skoog; PGRs, plant growth regulators.     

Integration of the rolA gene into the genome of the vigorous apple rootstock A2 reduced plant height and shortened internodes

Summary

The aim of this work was to dwarf the vigorous apple rootstock A2 by insertion of the rolA gene. To optimize conditions for a successful transformation, regeneration tests were carried out. The use of sucrose in the regeneration medium gave higher regeneration frequency than sorbitol in some cases and the shoot number per regenerated leaf was higher at 10 |j.M TDZ compared with 2.5 |j.M TDZ on the sucrose medium. Two transgenic clones, verified by PCR and Southern analysis, have been obtained on the sucrose medium together with 2.5 |j.M TDZ and 1.0 or 2.5 |j.M NAA and wounding by forceps. The two clones, named LAI and LA2, contained both the rolA and nptll genes. The results of in vitro rooting showed that LAI had a lower rooting percentage and a reduced root number per rooted shoot than the untransformed control shoots and the clone LA2 on the rooting medium containing 5 |JLM IBA. Growth analysis revealed that both transgenic clones had a reduced plant height and a shortened internode length compared with the control plants. However, the node number and the stem diameter were significantly larger for clone LAI than clone LA2 and the control plants.     

Molecular identification and antibiotic control of bacterial contamination in cultures of ginger (Zingiber officinale)

Zingiber officinale, Rosc., one of the major tropical spices in the world, belongs to the family Zingiberaceae. Bacterial contamination is a major constraint on the cryopreservation of in vitro shoot tip explants. The main objective of this study was to identify the nature of this bacterial contamination and its response to various antimicrobial agents (e.g. the antibiotics cephotaxime and streptomycin sulphate, or copper sulphate) for more effective control. The bacteria isolated from ginger plantlets were identified by amplification and sequencing of their 16S ribosomal DNA, followed by partial sequence analysis. Luteibacter yeojuensis was found in all contaminated cultures. L. yeojuensis is found mainly in soil and as a human pathogen. We believe this is the first report of L. yeojuensis contaminating in vitro cultures of ginger. Among the antimicrobial agents tested in the shoot multiplication medium [i.e. 1.0× Murashige and Skoog salts + 2.5 mg l^–l 6-benzylaminopurine + 3% (w/v) sucrose + 0.45% (w/v) Clarigar^TM], 100 mg l^–1 cephotaxime was most effective at reducing bacterial growth. It also gave the maximum number of shoots per shoot bud explant compared to the same medium supplemented with streptomycin sulphate, or CuSO₄, or control medium. No further bacterial contamination was observed when 8-week-old cultures were then subcultured on the same medium without added antibiotic or CuSO₄.     

Effects of media,carbon sources and cytokinins on shoot organogenesis in the Christmas tree Scots pine (Pinus sylvestris L.)

Summary

Significant effects of seven basal media and three carbon sources (sucrose, glucose and fructose) on the induction of adventitious buds from embryos of Pinus sylvestris L. were observed. Moreover, hyperhydricity of expiants and shoot regenerants was observed on basal media containing fructose, especially with half-strength Murashige and Skoog (MS), MS, and woody plant medium (WPM). Expiants grown on a Gresshoff and Doy (GD) medium with sucrose produced the highest frequency of regeneration (81%) and with no hyperhydricity observed of developing adventitious shoots. Among three cytokinins tested including BA, BPA, and TDZ (at four concentrations each), 5 μM BA resulted in the highest regeneration frequency and mean number of adventitious shoots per embryo. Shoot régénérants were elongated after transfer to a GD medium containing 2 g-l_–1 activated charcoal and no growth regulators. After one month, rooting was induced on 10% of expiants.     

多花指甲兰非共生萌发技术研究

多花指甲兰属于单轴分枝类兰花、无分蘖能力,种子在自然条件下萌发困难,自然更新能力差.研究采用人工授粉的种子,进行了非共生萌发研究.结果表明:110 d胚龄的种子萌发率高,种子萌发过程中原球体呈黄色并且无指状突起物的产生;种子萌发最佳培养基为MS+NAA 5.0 mg/L+BA 1.0 mg/L+活性碳0.6 mg/L+椰肉3.0 g/L,壮苗培养基以MS+NAA4.0 mg/L+BA 0.2 mg/L+活性碳0.6 mg/L为好,小苗用松树皮移栽效果好.该研究实现了多花指甲兰的快速繁殖,提高了其繁殖系数.     

Asymbiotic seed germination,mass propagation and seedling development of Vanda coerulea Griff ex.Lindl. (Blue Vanda): An in vitro protocol for an endangered orchid

The application of modern biotechnology for conservation of any endangered species requires an efficient in vitro regeneration protocol. In this study a reliable protocol was developed for in vitro seed germination, protocorm multiplication and subsequent plantlet regeneration of Vanda coerulea, an endangered orchid species. Among the four basal media evaluated for asymbiotic seed germination, Phytamax was found to be the best followed by Murashige and Skoog (MS). Phytamax was also found good for protocorm development. For protocorm like body (PLB) regeneration, protocorms were then further cultured on Phytamax media fortified with different phytohormones either individually or in combinations. The frequency of protocorm like body (PLB) regeneration significantly relied on kinds and concentrations of plant growth regulators used. A combination of 1-naphthaleneacetic acid (NAA) (5.36 μM) and 6-benzyle amino purine (BAP) (3.80 μM) was found to be suitable for maximum PLB regeneration. Healthy plantlets were induced from PLBs when cultured on same basal medium supplemented with activated charcoal (AC – 3.0 g/l). Plantlets with well developed leaves and roots were transplanted to pots filled with a mixture of charcoal, brick pieces and sphagnum moss and transferred to the greenhouse. This protocol will enable mass propagation and conservation of this exquisite orchid.     

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.