Acclimatization In Greenhouse Research Articles

Tissue Culture via Protocorm-like Bodies in an Orchids Hybrids Paphiopedilum SCBG Huihuang90.

This study successfully established an efficient in vitro propagation system for Paphiopedilum SCBG Huihuang90 via protocorm-like body (PLB) formation from seed-derived calluses, PLB proliferation and differentiation, root induction and greenhouse acclimatization. Furthermore, 1/2 Murashige and Skoog (MS) + 0.025 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) was suitable for the proliferation of PLBs, and 1/2MS + 10% coconut water (CW, v/v) + 0.5 g/L activated carbon (AC) was suitable for PLB differentiation. PLBs at different developmental stages required different kinds of sugars. This study provided a reference for research on the propagation techniques of other Paphiopedilum.

Aloe monticola Reynolds: A refugee of the mountains − contributing towards its conservation through in vitro propagation

In vitro micropropagation study of Aloe monticola Reynolds was conducted using offshoots in Murashige and Skoog (MS) media enriched with plant growth regulators (PGRs). Initiation experiment, carried out by culturing sterilized explants in full-strength MS media enriched with 0.0–1.0 mg/L benzyl amino purine (BAP) alone and in combination with 0.10 mg/L indole-3-butyric acid (IBA), resulted in 89 − 100 % healthy and live (i.e., initiated) explants in 9–29 days. Regeneration study, conducted by culturing initiated explants in full-strength MS media supplemented with 0.0–3.0 mg/L BAP alone and in combination with 0.50 mg/L IBA, showed that treatments enriched with 1.0–3.0 mg/L BAP in combination with 0.50 mg/L IBA yielded better shooting responses than the rest of the treatments. The rooting responses of the shoots were also tested by culturing in half-strength MS media enriched with 0.0–4.0 mg/L NAA alone and in combination with 0.25 mg/L IBA. Better rooting responses were observed in treatments supplemented with 1.0–4.0 mg/L NAA in combination with 0.25 mg/L IBA with two exceptions. The responses of A. monticola plantlets to primary and secondary acclimatization in greenhouse, nursery shade, and direct sunlight in coco peat, composted soil, and manured soil were excellent – with survival percentages of 90–98 %. The findings of this empirical research are important for developing refined protocol of in vitro micropropagation of this ecologically important but endangered plant.

Evaluation of the effect of different culture systems on photomixotrophic capacity during in vitro multiplication of pitahaya (Hylocereus undatus)

Pitahaya (Hylocereus undatus) micropropagation is an alternative for obtaining invigorated plants with high phytosanitary quality. The objective of this research was to evaluate different culture systems on photomixotrophic capacity during in vitro multiplication and their effect on survival in the acclimatization stage of H. undatus. Different temporary immersion systems (TIS) were used: temporary immersion bioreactors (TIB), Ebb-and-Flow bioreactor, Recipient for Automated Temporary Immersion (RITA), and semi-solid (SS) culture medium. At 60 d of culture, the percentage of explant response, developmental variables, chlorophyll and carotene content, stomatal index (SI), percentage of closed stomata (CS), PEP (Phosphoenolpyruvate) and Rubisco (Ribulose-1,5-bisphosphate carboxylase oxygenase) content and the percentage of survival in the greenhouse acclimatization stage were evaluated. The culture systems with the highest percentage of response were the SS culture medium (96%), Ebb-and-Flow (93%) and RITA (90%). The Ebb-and-Flow and RITA bioreactors showed the highest number of shoots, with 9.5 and 9.0, respectively, the greatest length, with 2.62 and 2.71 cm, the highest chlorophyll, with 0.30 and 0.28 mg g–1 FW, and carotenoid content, with 2.25 and 2.20 mg g–1 FW. The SS medium culture system showed the highest SI with (57%), while the TIB had the highest CS, with (85%). The Ebb-and-Flow and RITA culture systems showed the highest PEP content, with 15 and 18 nmol g–1 FW, respectively; whereas, for Rubisco content, no differences were found in the different culture systems. The Ebb-and-Flow and RITA culture systems presented the highest survival rate, with 99.6% during acclimatization. The use of Ebb-and-Flow and RITA bioreactors are a biotechnological alternative for in vitro propagation of H. undatus and guarantee high survival rates during acclimatization without going through a rooting stage.

An Efficient Protocol for In vitro Regeneration and Acclimatization of Banana (Musa spp.) cv. Grand Naine

Aim: The study was undertaken with a view to standardize a protocol for in vitro regeneration of Banana cv. Grand naine using shoot tip of sucker as an explant
 Methodology: In present study, explants sterilized with different sterilizing agents such as Tween-20 (1%), Bavistin (0.5-1%), Streptomycin sulphate (250 mg/L), Ascorbic acid (150 mg/L) + Citric acid (100 mg/L), HgCl2 (0.1%) and 70% ethanol.Sterilized explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of plant growth hormones for shoot initiation BAP alone (1.5, 2.0, 4.0, 6.0 mg/L) and BAP (3.0 mg/L) in combination with IAA and IBA (2.0 mg/L), elongation BAP (3.0 mg/L) and NAA (1.0, 1.5, 2.0 mg/L) and rooting IAA (1.0, 1.5 mg/L) and IBA (1.0, 1.5 mg/L). Primary and secondaryhardening was done in potting mixture containing autoclaved black soil: vermicompost: cocopeat (1:1:1) and garden black soil, cocopeat and red soil (1:1:1) respectively.
 Results: In present investigation 1% Bavistin (fungicide) showed maximum respond to prevent fungal contamination. Highest shoot initiation (100%) was observed on a MS medium fortified with BAP (1.5 mg/L). Maximum shoot length (10.7 cm) was recorded on a MS medium supplemented with BAP (3.0 mg/L) + NAA (2.0 mg/L) + Activated charcoal. Maximum root initiation was observed on half strength MS medium supplemented with IAA (1.5 mg/L). In vitro regenerated plantlets hardened on the mixture of autoclaved black soil: vermicompost: cocopeat (1:1:1). After 14 weeks In vitro plantlets transferred in green house for acclimatization where, 80% survival rate was recorded.
 Conclusion: Regeneration protocol was successfully standardized. Therefore, itcan be used for large scale propagation of healthy, disease and virus free planting material and In vitro propagation helps to meet higher demand of healthy planting material within shorter period.

Arbuscular Mycorrhizal Symbiosis Improves Ex Vitro Acclimatization of Sugarcane Plantlets (Saccharum spp.) under Drought Stress Conditions.

The symbiotic associations between arbuscular mycorrhizal fungi (AMF) and plants can induce drought stress tolerance. In this study, we evaluated the effect of Glomus intraradices, a mycorrhizal fungus, on the ex vitro development and survival of sugarcane plantlets subjected to drought stress during the acclimatization stage of micropropagation. In vitro obtained sugarcane plantlets (Saccharum spp. cv Mex 69-290) were inoculated with different doses of G. intraradices (0, 100, and 200 spores per plantlet) during greenhouse acclimatization. Sixty days after inoculation, plantlets were temporarily subjected to drought stress. We evaluated the survival rate, total chlorophyll, total protein, carotenoids, proline, betaine glycine, soluble phenolic content, and antioxidant capacity every 3 days for 12 days. Symbiotic interaction was characterized by microscopy. Our results showed that the survival rate of inoculated plants was higher in 45% than the treatment without mycorrhizae. Total chlorophyll, protein, proline, betaine glycine content, and antioxidant capacity were increased in AMF inoculated plants. The soluble phenolic content was higher in non-inoculated plants than the treatment with mycorrhizae during the drought stress period. Microscopy showed the symbiotic relationship between plant and AMF. The early inoculation of 100 spores of G. intraradices per sugarcane plantlet during the acclimatization stage could represent a preconditioning advantage before transplanting into the field and establishing basic seedbeds.

Optimizing Growth Conditions for Green Grass Jelly (Cyclea barbata Miers) Derived in Vitro Culture: Greenhouse Acclimatization and Field Growth

Cyclea barbata Miers, a species within the Menispermaceae family, holds significance in medicinal and beverage applications. In vitro propagation is a common practice for conserving this plant; however, a comprehensive investigation of its growth characteristics within controlled greenhouse and natural field environments is essential. This study aims to determine the optimal planting media for acclimatization and the subsequent growth of Cyclea barbata Miers propagated from in vitro cultures. The experimental design encompasses two phases: 1) greenhouse acclimatization and 2) field growth. Various planting media, including combinations of sterile soil, manure, and husk charcoal, were rigorously evaluated during the acclimatization phase. Following the acclimatization period, the plants were transferred to the field. Multiple parameters were assessed: plant growth percentage, branch and node counts, leaf dimensions (length and width), and leaf wet and dry weights. Our findings reveal that a planting medium comprising sterile soil, manure, and husk charcoal provided the best performance growth outcomes during acclimatization. Furthermore, we observed that plant age exerts a discernible influence on the growth dynamics of Cyclea barbata Miers.

STANDARDIZATION OF MICROPROPAGATION TECHNIQUE OF MULBERRY (MORUS ALBA L.)

A standardization of protocol for rapid multiplication of mulberry (Morus alba L.) was established in vitro using nodal explant excised from hydroponically grown mature mulberry plants. Shoot initiation was induced on Murashige and Skoog (MS) medium supplemented with different concentrations of (BAP) 6- Benzyl amino purine. Effect of different concentration of 6- Benzyl amino purine was studied on multiplication of shoot, revealed that elongated shoots (2-3 cm) were cultured on half strength MS medium supplemented with (IBA) indole-3-butyric acid. Higher percentage of shoot initiation (90%) was observed on MS medium supplemented with 2 mg/l 6-bezyl amino purine. Initiated shoots gave a higher average number of shoots and elongation was observed when it was subcultured on MS medium supplemented with 1.4 mg/l 6-bezyl amino purine. A maximum percentage of root formation (70%) was observed when elongated shoots were subcultured on half strength MS medium containing 1.3 mg/l indole-3-butyric acid. Rooted plants transferred to pots containing sterile FYM, garden soil and sand (1:1:1) for acclimatization in green house showed (75%) survival capacity.

Assessment of Plant Growth Regulators and Carbon Sources on the Germination and Growth Process of Dandelion (Taraxacum officinale G.H. Weber ex Wiggers) under In Vitro Conditions

There are several studies on the medicinal properties of dandelions (Taraxacum officinale), but few studies are aimed at understanding the in vitro germination process of this plant to improve its propagation. This research was focused on studying in vitro seed germination and development of seedlings under different carbon sources, glucose (GLU) or sucrose (SUC) and its concentrations (1.0–5.5%). Additionally, the effect of supplementation with plant growth regulators (PGRs) was studied, measuring the germination capacity, uncertainty and synchrony. Germination was promoted under low carbon source concentrations (≤2.3%), whereas higher concentrations (≥3.2%) had a detrimental effect on this process. GLU allowed the final germination percentages to be slightly better than SUC. Uniformity and synchrony values improved with the presence of PGRs. Results suggested that the best condition to assess T. officinale seed germination is in a medium containing GLU 2.3%, 0.5 mg/L 1-Naphthaleneacetic acid (NAA) and 0.5 mg/L 6-Benzylaminopurine acid (BAP). After germination, the best condition for optimal growth of T. officinale seedlings was 1.0% SUC supplemented with 0.225 mg/L of NAA and 3.0 mg/L of BAP for initial shoot development. The survival rate was 97% after greenhouse acclimatization. This new method of germination was implemented for the massive propagation of T. officinale for further medicinal studies.

Correction to: Synergistic effect of cytokinins and auxins enables mass clonal multiplication of drumstick tree (Moringa oleifera Lam.): a wonder

The synergistic effect of plant growth regulators on axillary bud proliferation for mass clonal multiplication of Moringa oleifera Lam. (vern. drumstick) has been assessed for the first time. Treatment of decoated seeds with 1% (w/v) Bavistin for 60 min, 0.33% (w/v) streptocycline for 30 min, and 0.1% (w/v) HgCl2 for 3.5 min resulted in complete removal of the surface contaminants. Maximum seed germination (89.13%) was obtained on quarter-strength Murashige & Skoog (MS) medium. Culture of nodal segments on MS + 6-benzyladenine (BA) at 3 mg L−1 resulted in multiple shoot proliferation with ~ 18 shoots per explant. All combinations of indole-3-acetic acid (IAA) + kinetin (Kn) resulted in elongated shoots, while only lower concentrations of BA (0.5 mg L−1), along with IAA (0.5 to 2 mg L−1), or Kn (0.5 to 5 mg L−1), showed significant synergy in the shoot morphogenesis. In addition, the maximum (100%) rooting efficiency was attained on half-strength MS medium supplemented with different concentrations of IAA and indole-3-butyric acid (IBA). The rooted plants were successfully established in the greenhouse for acclimatization. Clonality of the raised plants was assessed using 15 random primers of Operon® technologies (OPT and OPF series), and eight primers resulted in significant amplification with distinct, identical, and reproducible bands that confirmed clonality of the micropropagated plants. The present study provides a comprehensive analysis of the synergistic effect of plant growth regulators (PGRs) on in vitro shoot regeneration and proliferation for clonal mass multiplication disease-free plantlets, which can be utilized to maximize the yield of healthy and genetically identical plants of drumstick tree, which is considered to be a miracle multipurpose tree.

In vitro and ex vivo vegetative propagation and cytokinin profiles of Sceletium tortuosum (L.) N. E. Br.: a South African medicinal plant

Sceletium tortuosum is a South African protected species with tremendous value in traditional and modern medicine. The plants’ mesembrine-type alkaloids are potential therapeutics for a plethora of psychological, neurological and inflammatory disorders. In our in vitro and ex vivo studies, vegetative propagation and growth of this species were investigated. Cytokinin (CK) profiles were also explored. Shoot multiplication was induced on Murashige and Skoog (MS) medium supplemented with 2.5 µM indole-3-butyric acid (IBA). In vitro-generated shoots were inoculated on MS medium supplemented with 0, 2.5, 5.0 and 10.0 µM IBA or indole-3-acetic acid (IAA). Optimal rooting (55%), mean number of roots (3.80 ± 0.83) and new leaf pairs (4.65 ± 0.67) were achieved by 10.0 µM IBA. After greenhouse acclimatization, 45–90% of plantlets survived. All ex vivo shoot cuttings rooted well (90–100%). The highest mean number of roots (11.20 ± 1.37) and root length (57.18 ± 3.85 mm) were obtained by 5.0 µM IBA. Although spontaneous rooting was observed in both experiments, auxins enhanced multiple growth parameters. Cytokinin analyses of tissue-cultured (auxin-treated) and greenhouse (untreated) plants revealed higher cytokinin levels in vitro. These investigations provide rapid and efficient propagation techniques for Sceletium tortuosum which will be valuable to conservationists and pharmaceutical companies. Plant tissue culture and cuttings enabled rapid propagation of Sceletium tortuosum. Exogenous plant growth regulators were not essential for shoot multiplication, flowering and rooting. Auxins effectively improved growth parameters.

Effect of commercial plant growth regulator on the growth of dragon fruit (Hyolocereus sp) cuttings under greenhouse condition

Information is limited on the effects of commercial plant growth regulators (PGRs) on dragon fruit. Most studies on rooting effects on dragon fruit cuttings employed very high doses of plant growth regulators (PGRs), which are not deemed practical for greenhouse and field applications. This study was initiated to evaluate the responses of four genotypes of dragon fruit (‘Royal Red’, ‘Moroccan Red’, ‘Mexican White’, and ‘Hawaiian White’) during greenhouse acclimatization to diluted concentrations of commercial PGRs that are readily available in the market. A single factorial experiment on genotype effects on root and shoot responses, without PGR, was conducted. A second experiment with a 4x3x2 completely randomized design, with four genotypes, three diluted commercial PGR formulations and two exposure times as factors was also done. Root length, number of roots, number of days to first shoot emergence and number of new shoots were measured after 50 days of acclimatization in the greenhouse. In the single factorial experiment, only ‘genotype’ had an effect on number of new shoots (P<0.05). In the multifactorial experiment, all factors tested did not have significant interaction effects. However, the main effect of ‘genotype’ on number of new shoots and number of days to first shoot emergence was also significant (P<0.05). The lack of significant effect of other factors and their interactions on most growth responses suggest that it is difficult to resolve differences among the four genotypes using highly diluted commercial PGR formulations after a 50-day observation period. However, our data still suggest that the ‘Red’ genotypes are more superior than the ‘White’ genotypes in terms of shoot growth.

In Vitro Micropropagation of Industrially and Medicinally Useful Plant Aloe trichosantha Berger Using Offshoot Cuttings.

This study was aimed to develop in vitro micropropagation protocol of Aloe trichosantha Berger using offshoots as explants. MS media supplemented with plant growth regulators helped explants develop shoots within about 14 to 17 days. The mean number of days to shooting has decreased from 16.8 ± 0.8 with 0.5/0.5 mg/L BAP/NAA supplement to 15.5 ± 0.5 with 2.0/0.5 mg/L BAP/NAA. While the mean shoot number has increased with increasing the concentration of BAP supplements, the reverse was true with mean shoot lengths, whereas supplement of 2.0/0.5 mg/L BAP/NAA has generated significantly more shoots (17 ± 3.8), and longer shoots were produced with the addition of 0.5/0.5 and 1.0/0.5 mg/L BAP/NAA. In regard to rooting, though higher concentrations of NAA have resulted in quick rooting, the rooting performance in terms of mean number and length of roots was better with low concentrations. All the plantlets subjected to greenhouse acclimatization in cocopeat have survived. Secondary acclimatization in composted and manured soil media has also resulted in 93 to 95% survival rate. Lighting conditions (nursery shade or direct sunlight) of secondary acclimatization did not lead to any difference in the survival rate of the plantlets.

In vitro Propagation of Two Grapevine (Vitis vinifera L.) Cultivars under Conditions of Salt Stress

This study was aimed to investigate salt tolerance of two grapevine cultivars, ‘Chenine Blanc’ and ‘Canonannon’ through in vitro propagation on medium containing different concentrations of NaCl. Single-node shoots were cultured on MS with 1.0 mg/l BAP in combination with 0.1 mg/l IBA and containing 0.25, 0.50, 0.75, 1.00 or 1.50% NaCl. NaCl free medium was used as control. Shoots of both cultivars were cultured on the same MS containing 0.25, 0.50, 0.75 or 1.00% CaCl2 to reduce hyperhydricity. The shoots were transferred to rooting medium followed by acclimatization in greenhouse. Number and length of shoots and roots, number of leaves and nodes, length of nodes, fresh and dry weight of roots and shoots decreased significantly in consistent trend as the concentration of NaCl increased. ‘Canonannon’ cultivar was found to be significantly more tolerant to NaCl than ‘Chenine Blanc’ in all parameters. The lowest percentage of hyperhydric shoots were obtained on medium containing 0.25% CaCl2. Therefore, ‘Canonannon’ cultivar can be planted in relatively saline soils as it is more tolerant to salt than ‘Chenine Blanc’.
 Plant Tissue Cult. & Biotech. 30(1): 47-56, 2020 (June)

Synergistic effect of cytokinins and auxins enables mass clonal multiplication of drumstick tree (Moringa oleifera Lam.): a wonder

The synergistic effect of plant growth regulators on axillary bud proliferation for mass clonal multiplication of Moringa oleifera Lam. (vern. drumstick) has been assessed for the first time. Treatment of decoated seeds with 1% (w/v) Bavistin for 60 min, 0.33% (w/v) streptocycline for 30 min, and 0.1% (w/v) HgCl2 for 3.5 min resulted in complete removal of the surface contaminants. Maximum seed germination (89.13%) was obtained on quarter-strength Murashige & Skoog (MS) medium. Culture of nodal segments on MS + 6-benzyladenine (BA) at 3 mg L−1 resulted in multiple shoot proliferation with ~ 18 shoots per explant. All combinations of indole-3-acetic acid (IAA) + kinetin (Kn) resulted in elongated shoots, while only lower concentrations of BA (0.5 mg L−1), along with IAA (0.5 to 2 mg L−1), or Kn (0.5 to 5 mg L−1), showed significant synergy in the shoot morphogenesis. In addition, the maximum (100%) rooting efficiency was attained on half-strength MS medium supplemented with different concentrations of IAA and indole-3-butyric acid (IBA). The rooted plants were successfully established in the greenhouse for acclimatization. Clonality of the raised plants was assessed using 15 random primers of Operon® technologies (OPT and OPF series), and eight primers resulted in significant amplification with distinct, identical, and reproducible bands that confirmed clonality of the micropropagated plants. The present study provides a comprehensive analysis of the synergistic effect of plant growth regulators (PGRs) on in vitro shoot regeneration and proliferation for clonal mass multiplication disease-free plantlets, which can be utilized to maximize the yield of healthy and genetically identical plants of drumstick tree, which is considered to be a miracle multipurpose tree.

In vitro culture and greenhouse acclimatization of Oncidium varicosum (Orchidaceae) with microorganisms isolated from its roots

Abstract Sustainable production of orchids may be achieved with the use of symbiotic microorganisms that improve resistance of plants to pests and diseases and reduce the need of substrate fertilization. In this study, one fungus (F1), one yeast (Y1) and one gram-positive bacteria (B+) were isolated from the roots of the orchid Oncidium varicosum and used in the cultivation of protocorms and plantlets obtained from seeds of this species. Cultivation of protocorms in Murashige and Skoog (MS) medium enriched with sucrose and inoculated with microorganisms did not result in better development of protocorms. However, the use of plantlets in the rooting phase in MS medium without sucrose and inoculated with B+ resulted in plantlets with more leaves and roots and longer shoots and roots, suggesting synergism between microorganism and plant when sucrose is absent. The major benefits of co-cultivating plantlets and these microorganisms were observed in the acclimatization in the greenhouse. Plantlets obtained with oat meal agar medium and B+ showed the highest pseudobulb diameter, number of roots, height and fresh weight among treatments. We concluded that the application of rooting-derived microorganisms in the in vitro culture and plantlet acclimatization of Oncidium varicosum is beneficial.

Influence of Continuous Spectrum Light on Morphological Traits and Leaf Anatomy of Hazelnut Plantlets.

Light spectra influence growth, development, and quality of plants and seedlings, that is one of the main aspects engaging the interests of private and public researchers and nursery industries. Propagation of hazelnut (Corylus avellana L.), which in the past has been held in low consideration because of the widespread use of rooted suckers directly collected in the field, today is taking on increasing interest due to the strong expansion of hazelnut cultivation. In order to improve the quality of plants and seedlings in greenhouse acclimatization, the effects of light emitting diodes (LED) lights during the ex vitro growth of two hazelnut cultivars (Tonda di Giffoni and Tonda Gentile Romana) were investigated. Plantlets were maintained in a growth chamber and exposed to three different continuous spectrum LED systems as a primary source of illumination and to fluorescent lamps used as control. LEDs differed in the percentage of some wavelength ranges in the spectrum, being AP673L rich in green and red wavelengths, NS1 in blue and green light, G2 in red and far red wavelengths. After a 4-week experimental period, morphometric, biochemical, and histological analyses were carried out. Shoot and leaf growths were influenced by LEDs more than by fluorescent lamps in both cultivars. G2 positively affected biomass increment more than the other LEDs, by inducing not only cell elongation (increase in shoot length, new internodes length, leaf area) but also cell proliferation (increase in new node number). G2 exposure had negative effects on total chlorophyll content but positively affected synthesis of flavonoids in both varieties; therefore, plants grown under this LED showed the lowest nitrogen balance index. Leaf morpho-anatomical analyzed traits (thickness, palisade cell height, number of chloroplasts, number of palisade cells), were influenced especially by G2 and, to a less extent, by NS1 light. Significant differences in some parameters were observed between the two cultivars in response to a same light source. The results obtained underline the importance of light modulation for hazelnut, providing useful information for ex vitro growth of hazelnut plantlets.

In Vitro Propagation and Acclimatization of Dragon Tree (Dracaena draco)

In this study, an efficient in vitro procedure was developed for bud induction, rooting of developing shoots and greenhouse acclimatization of young plantlets of dragon tree (Dracaena draco). Effects of media (S1 (1 mg/L KIN and 1 mg/L NAA), S2 (3 mg/L KIN and 1 mg/L IAA), S3 (1 mg/L BAP and 2 mg/L IBA) and S4 (1 mg/L BAP and 1 mg/L NAA)) on shoot induction and media (R1 (0 mg/L IBA), R2 (0.5 mg/L IBA), R3 (1 mg/L IBA), and R4 (2 mg/L IBA)) on root induction were examined in order to find optimal plant hormone concentrations for efficient Dracaena draco dormant bud development and subsequent rooting. The best shoot induction and rooting media were S1 and S2, and R3 and R4, respectively. Dormant buds from one-year-old Dracaena draco plants submitted to this in vitro procedure allowed successful recovery of up to 8 individuals per explant used. In vitro grown plants were successfully acclimated in the greenhouse. The potential of this in vitro procedure for multiplication of this endangered tree is discussed in this report.

In vitro desiccation tolerance of the epiphytic Ghost Orchid, Dendrophylax lindenii (Lindl.) Benth x. Rolfe

The Ghost Orchid, Dendrophylax lindenii (Lindl.) Benth x. Rolfe, is a rare and endangered epiphytic orchid native to south Florida and Cuba. The orchid is considered difficult to propagate under greenhouse conditions, requiring high humidity and low air movement. In contrast, the orchid’s native habitat seasonally dries out with decreased precipitation and humidity. This suggests some level of desiccation tolerance. Ghost Orchid plants were assessed for potential desiccation tolerance and ability to recover from desiccation stress under in vitro conditions. In vitro-derived plants were placed into sterile baby food jars and transferred to chambers maintained at 10% relative humidity, which is extremely low compared to relative humidity levels (40–100%) recorded under natural field conditions. Plants were removed every week for 4 wk and recovered on P723 medium supplemented with banana powder for 4 wk. Data were collected at the initiation of the experiment, after the desiccation periods, and after 4 wk in vitro recovery. Ghost Orchid plants demonstrated extremely high desiccation tolerance. Even after 4 wk desiccation, plant survival was observed at 79.2% after recovery. Desiccated plants exhibited significant decreases in tissue water potential (− 18.44 MPa), fresh weight (65.5% loss), and water content (14.2%); however, high plant survival was still observed under these conditions similar to poikilohydric plants. Overall, the Ghost Orchid demonstrated high desiccation tolerance, which should be considered for future greenhouse culture and for its application in the direct field establishment of in vitro-derived plants without greenhouse acclimatization.

Ex vitro establishment and macropropagation of cassava (Manihot esculenta ‘Valencia’) to obtain disease-free rooted plants

A common practice during cassava cultivation in Costa Rica is the use of stem cuttings coming from farmers' plantations as vegetative seeds. This system has widely spread diseases all over the country, especially the phytoplasm associated “Frog Skin” disease. At Tecnologico de Costa Rica we developed a protocol for rapid multiplication of disease-free plantlets. Terminal buds obtained from in vitro plants were grown in a MS (1962) medium under four different light treatments: 100% blue light (465 nm), 100% red light (630 nm), 50-50% red-blue light and white fluorescent light (as control), all of them with a light intensity (PPFD) of 70 μmol m-2 s-1. The best plantlets for acclimatization were obtained with blue light, which resulted in thicker stems and shorter internodes. In a second experiment, in vitro plants were transferred to the greenhouse for acclimatization, where five different substrates were evaluated: coco-coir, peat moss with perlite (75-25), mix of coco-coir with peat, sterile sand and sterile mix of soil+sand. The percentage of successfully acclimated plants was above 90% for those grown with coco-coir and peat moss, 86% for mix coco-coir+peat moss, 82% for soil+sand and 60% for sand-grown plants. Acclimatized plants were taken to a greenhouse and grown in pots with coco-coir during 8 weeks; after this time, plants became fully developed. Then cuttings of 10 cm length were obtained from the top of the plant and rooted using IBA (indolebutyric acid) in four concentrations: 0, 500, 1000 and 1500 ppm. The higher number and length of roots resulted in those cuttings treated with 1000 ppm. After three weeks, the rooted cuttings were taken successfully to the field, with a mortality of 4%.

In vitro propagation of Epipactis flava Seidenf., an endangered rheophytic orchid: a first study on factors affecting asymbiotic seed germination, seedling development and greenhouse acclimatization

Epipactis flava Seidenf., is an endangered Thai rheophytic orchid that has decreased rapidly throughout its natural habitat and urgently requires conservation using in vitro techniques. Effect of pollination types (self- and cross-pollination) and seed from different capsule ages (2, 4, 6 and 8 weeks after pollination) on asymbiotic seed germination efficiency was performed on semi-solid VW medium supplemented with 150 mL/L coconut water and 50 g/L potato extract. Highest germination rate was 79.2% with definite rate of protocorm developmental stage 5 at 24.0% using seeds from 6 week old cross-pollination capsules. Anatomical investigation revealed young seed has been formed at 6 weeks old capsules. Identical seeds were then cultured on semi-solid and liquid systems of five different media including modified-VW, MS, BM, MM and KC for 2 months. Results showed that semi-solid and liquid VW medium promoted the highest seed germination rates at 70.2% and 70.4%, respectively, with the highest definite rate of protocorm developmental stage 5 at 54% found on semi-solid BM medium. In vitro seedlings were transferred to culture on both semi-solid and liquid VW and MS media for 2 months. Higher growth parameters, as indicated by shoot number and fresh weight, were obtained on liquid MS medium. Clumps of plantlets containing 5–8 shoots were acclimatized and transplanted into different potting media for 3 months. Survival percentages in all tested substrates were recorded at nearly 100% with no significant differences, while 63–68% of the living plantlets produced new shoots.