In vitro propagation and field establishment of Eulophia cullenii (Wight) Bl., a critically endangered orchid of Western Ghats, India through culture of seeds and axenic seedling-derived rhizomes

An efficient protocol has been devised for the propagation and field establishment of Eulophia cullenii (Wight) Bl., a terrestrial orchid having ornamental potentialities, and is critically endangered in Western Ghats, India. Seeds extracted from 60–90-d-old capsules germinated in ½ MS, ¼ MS, Knudson C, or Mitra liquid medium developed into 1.4–2.5-mm-diameter protocorms in 60 d. Supplementation of organic additives like coconut water, peptone, yeast extract, and casein acid hydrolysate (CH) significantly enhanced protocorm growth. Upon subculture onto agar-gelled Mitra medium fortified with 0.05% CH, 56% of protocorms regenerated into shoots through the formation of linear mini-rhizomes. The regenerated shoots grew vigorously in ½ MS, producing new rhizomes. Mature rhizomes from axenic seedlings produced maximum (13?±?1.4) shoots/whole rhizome in ½ MS fortified with 44.4 μM 6-benzylaminopurine (BAP), in 120–150 d. Horizontal and longitudinal halves of the rhizome also gave multiple shoots (6–8.5) in the presence of 44.4 μM BAP. Shoots or shoot clumps sub-cultured onto ½ MS basal medium produced roots followed by rhizomes in 60–150 d. Seedlings with mature rhizomes showed 70% establishment in the nursery and added a new rhizome at the end of one growth cycle. An average of 70.6% of the rhizomes originating from seedlings during the second growth cycle sprouted to produce new shoots, when planted in the native localities. Asymbiotic germination and cloning through rhizomes thus can provide a large number of vigorous plants of E. cullenii for ornamental exploitation as well as eco-restoration, if rhizome as storage organ is ensured in the propagule.     

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.     

In vitro propagation of Dendrobium aphyllum (Orchidaceae)—seed germination to flowering

This communication describes asymbiotic seed germination, protocorm development, micropropagation and flowering in in vitro and hardened seedlings of Dendrobium aphyllum (Roxb.) C.E.C. Fischer. Effects of four culture media viz., Murashige and Skoog (MS); Phytamax (Sigma Chemical Co. USA; PM); Mitra et al. (M) and Knudson ‘C’ (KC), 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D), peptone and activated charcoal were studied on seed germination and protocorm development. Maximum germination (97 %) was recorded in PM basal medium. Peptone (2.0 gl^?1) remarkably enhanced germination percentage (100 %), vigorous growth, high survival and subsequent development of protocorms, while in activated charcoal the response was not encouraging. BAP improved germination percentage, however, 2,4-D showed noticeably low seed germination. The morphogenetic response of protocorms and nodal segments of in vitro raised seedlings varied depending on type of explants and concentrations and combinations of plant growth regulators used. Stout root system was induced in 1/2PM + 0.5 mgl^?1 IAA. Approximately 10 % of the in vitro raised plants (4–5 cm) with 3–4 leaves flowered in vitro irrespective of flowering season. The well-rooted plants showed 80 % survival under green house conditions and flowering was noticed after 5–6 months in 10 % of hardened plants.     

An efficient in vitro propagation,antioxidant and antimicrobial activities of Aphyllorchis montana Rchb.f.

An in vitro plant regeneration protocol was successfully established in Aphyllorchis montana, a saprophytic achlorophyllous orchid by culturing immature seeds. Among the six basal media evaluated for seed germination, BM-1-Terrestrial Orchid medium was found to be the best followed by Knudson C medium. Half-strength BM-1-Terrestrial Orchid medium was supplemented with different growth regulators either individually or in combinations for multiplication of shoots induction. Of the five cytokinins tested, thidiazuron at 6.8 μM was found to be most effective for multiple shoot induction yielding 17.24 ± 0.27 shoots after 10 weeks of culture. Addition of low concentration of α-Naphthaleneacetic acid (1.3 μM) in MS medium supplemented with the cytokinin thidiazuron (6.8 μM) favored shoot multiplication. The addition of organic additives to the medium containing thidiazuron enhanced the number of regenerated shoots. The plantlets were acclimatized, and the survival rate was 100%. Screening of the antioxidant and antimicrobial activities and estimation of total phenolics and flavonoid content of methanolic extracts of micropropagated plants were also carried out and compared with that of the wild-grown plants. In all the tests, methanolic extract from wild-grown plants showed higher antioxidant, antimicrobial activity, total phenolics and flavonoid content than in vitro propagated plants.     

In vitro symbiotic seed germination of South Indian endemic orchid Coelogyne nervosa

Study on the dependence of orchids on fungi for seed germination and seedling development provides a mean for understanding the role of fungi in the orchid development process. The epiphytic orchid Coelogyne nervosa endemic to south India is exploited in an unsustainable manner for its therapeutic value. So a protocol for symbiotic seed germination was established for C. nervosa. We isolated a fungus by plating mycorrhizal root discs of the terrestrial orchid Eulophia epidendreae and identified it as Epulorhiza sp., by sequencing the internal transcribed spacer (ITS) regions of the ribosomal RNA gene. Germination of C. nervosa seeds was higher when inoculated with Epulorhiza sp. Uninoculated seeds of C. nervosa ceased to develop soon after the initiation of germination, and the embryo failed to rupture the seed testa. The isolated fungal hyphae entered the germinating seeds either through the pores in-between the integuments, or through the rhizoids. After the fungal establishment (peloton formation) in embryonic cells, the embryo transformed into a protocorm and after 45 days, 66% of the germinated seeds were transformed into protocorms. Nevertheless, promeristem formation occurred only after fungal association. Sixty-three percent of the protocorms developed their first leaf by 90 days and 62% of these produced a second leaf by 120 days after fungal inoculation. All the seedlings in green leaf stage produced roots and contained fungal pelotons. Our results suggest that the Epulorhiza sp. could be successfully used in the in vitro production of C. nervosa for their reintroduction into its natural environment.     

The Effect of Seed Cryopreservation on the Development of Protocorms by the Hybrid Orchid Bratonia

The aim of this work was to study the influence of storage in liquid nitrogen on the viability of seeds of the hybrid orchid Bratonia and further development of its protocorms in vitro. Seeds were frozen in ampoules by direct immersion in liquid nitrogen and stored in the cryobank for a month. The germination rates of cryopreserved and control (nonfrozen) seeds did not differ and remained as high as 100%. The protocorms derived were cultured on the agar-solidified Murashige and Skoog nutrient medium (MS), half-strength MS and Knop media and also in Morel liquid medium. During the first 45 days of culturing, protocorms derived from cryopreserved seeds grew faster than control protocorms on the MS and half-strength MS media but, at longer culturing (496 days), the size of control protocorms was significantly larger. After 639 days of culturing, there was no difference in the amount of perished, budding, and newly formed protocorms obtained from cryopreserved and control seeds, except half-strength MS medium where the number of budding protocorms in the case of cryopreserved seeds was a little greater than in the control treatment. After seed cryopreservation, the frequency of budding and newly formed protocorms was greater on the agarized MS and in liquid Morel media. Cryopreservation had little effect on the subsequent growth of protocorms in vitro. The preferable nutrient media for culturing the protocorms have been suggested.     

Storability, post-storage conversion and genetic stability assessment of alginate-encapsulated shoot tips of monopodial orchid hybrid Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl.

An efficient short-term storage system of synthetic seeds, produced using in vitro shoot tips of the monopodial orchid hybrid Aranda Wan Chark Kuan ‘Blue’ × Vanda coerulea Grifft. ex. Lindl. (AV), was developed. In vitro shoot tips (3–4 mm) were successfully encapsulated, resulting in uniform spherical beads (capsules), using 3 % sodium alginate with 75 mM CaCl₂·2H₂O. Maximum (~100 %) conversion (into plantlets with shoot and root) of capsules (or synthetic seeds) was achieved on quarter-strength Murashige and Skoog regrowth medium, while full-strength MS medium was required for effective conversion of non-encapsulated shoot tips. The capsules showed distinct difference in their response to temperature during storage. The conversion efficiency declined upon storage duration at both 4 and 25 °C, with those stored at 25 °C being more tolerant to storage. Capsules stored at 4 °C had rapid deterioration and faced complete death within 160 days while those stored for 200 days at 25 °C showed relatively high conversion (71.6 %). An inter-simple sequence repeats fingerprinting approach, employed on indiscriminately chosen plantlets from converted capsules (following 4 and 25 °C of storage), ensured the post-storage genetic stability.     

In vitro propagation of temperate Australian terrestrial orchids: revisiting asymbiotic compared with symbiotic germination

Using a common temperate herbaceous terrestrial orchid from Australia (Caladenia latifolia) this study investigated 19 asymbiotic media variations comprising four commonly used basal media [half‐strength Murashige and Skoog (½MS), Knudson C (KC), Pa5, and Vacin and Went (VW), with combinations of the plant growth regulators 6‐benzylaminopurine (BA) and α‐naphthalene acetic acid (NAA) or coconut water (CW) and compared their performance with germination on a standard symbiotic germination medium, oatmeal agar (OMA). Percentage germination of seeds every 2 weeks for a total of 8 weeks (five replicates per treatment), time to germination, and growth and development phases in seedlings were recorded. ½MS with 5% (v/v) fresh CW delivered 93% germination, with seedling vigour and development indistinguishable from OMA (95% germination). The same protocol was applied to a further ten species (including the endangered Caladenia huegelii), demonstrating high asymbiotic germination performance (60–93%) across a wide phylogenetic range of terrestrial orchid species. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 556–566.     

In vitro propagation of <Emphasis Type="Italic">Epipactis flava</Emphasis> 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.     

High frequency early in vitro flowering of <Emphasis Type="Italic">Dendrobium</Emphasis> Madame Thong-In (Orchidaceae)

We have successfully developed a method to induce early in vitro flowering of the self-pollinated seedlings of a tropical orchid hybrid, Dendrobium Madame Thong-In. Transition of vegetative shoot apical meristem to inflorescence meristem was observed when young protocorms were cultured in modified KC liquid medium. In contrast, protocorms cultured on Gelrite-solidified medium only produced axillary shoots and roots. CW was required to trigger the transitional shoot apical meristem and BA enhanced inflorescence stalk initiation and flower bud formation. However, normal flower development was deformed in liquid medium but developed fully upon transferring to two-layered (liquid over Gelrite-solidified) medium. Under optimal condition, in vitro flowering was observed about 5 months after seed sowing. Segregation of flower colours was observed in these seedlings and seedpods formed upon artificial pollination of the in vitro flowers.     

In vitro shoot regeneration from leaf explants of Echinops kebericho: an endangered endemic medicinal plant

Echinops kebericho is a critically endangered endemic medicinal plant of Ethiopia. It is threatened due to over harvesting of its roots for medicinal purposes and from poor seed viability. This study aimed to develop a protocol for in vitro shoot regeneration from leaf explants of E. kebericho. The seeds were sterilized using ethanol followed by Clorox or calcium hypochlorite. Shoots from the germinated seeds were cultured on Murashige and Skoog (MS) medium containing different concentrations of α-naphthalene acetic acid (NAA) and 6-benzyl amino purine (BAP). Young leaves were cultured on MS medium containing different concentrations of BAP and NAA for shoot regeneration. For shoot multiplication, shoots were excised and cultured on MS medium containing different concentrations of BAP or kinetin (KIN) and NAA. The highest mean number of initiated shoots (4.00 ± 0.57) with 100% shoot induction was obtained on medium containing 1.0 mg/L BAP and 0.2 mg/L NAA. The highest shoot regeneration (33%) and shoot number (2.13 ± 0.06) were obtained on MS medium containing 2.0 mg/L BAP and 0.5 mg/L NAA. Medium containing 1.0 mg/L KIN and 0.2 mg/L NAA produced the highest number of shoots (4.67 ± 0.33) per explant. This protocol can be used for genetic improvement and conservation of this endangered species.     

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.     

Cryopreservation of Cymbidium eburneum Lindl. and C. hookerianum Rchb. f., two threatened and vulnerable orchids via encapsulation–dehydration

A successful cryopreservation protocol for the long-term conservation of protocorms of two threatened and vulnerable orchids, Cymbidium eburneum Lindl. and Cymbidium hookerianum Rchb. f., was developed using encapsulation–dehydration. Protocorms were osmoprotected in liquid Murashige and Skoog medium (MS) containing 0.7 M sucrose for 20 h at 25?±?2°C on a rotary shaker, and incorporated into an encapsulation matrix [consisting of 3% (w/v) sodium alginate and 100 mM CaCl₂]. The encapsulated protocorms, which were desiccated in a laminar airflow cabinet for 6 h, were able to withstand cryostorage in liquid nitrogen. Maximum regeneration into complete plantlets (72% for C. eburneum and 70% for C. hookerianum) of the cryostored, encapsulated protocorms was obtained using MS medium containing 3% sucrose and 0.8% agar. Using this protocol of cryopreservation, long-term preservation for ex situ conservation of these two threatened orchids can be accomplished.     

In vitro conservation and asymbiotic propagation of Coelogyne flaccida (Lindl.): A threatened orchid

Asymbiotic seed germination of Coelogyne flaccida varied with the capsule stage and the culture medium used for germinating seeds. The capsules were harvested at two different stages of development. The seeds were cultured on three asymbiotic orchid seed germination defined and undefined media, i.e. Mitra (M) medium, Murashige and Skoog (MS) medium and potato dextrose agar (PDA) medium. The seeds obtained from undehisced green capsules germinated with a maximum germination percentage (84.50 ± 0.33%) on M medium followed by MS and PDA medium. The effect of cytokinins, such as 6-benzylaminopurine and furfurylaminopurine and the synthetic auxin α-naphthalene acetic acid, on seed germination was also assessed. Simultaneously, in vitro multiplication using protocorms as explants was also studied. The effect of organic growth supplements, such as banana homogenate (BH, 25, 50, 75 g l^? 1) and peptone (P, 1.0, 1.5, 2.0 g l^? 1), was tested on the de novo formation of protocorm-like bodies (PLBs), development of the maximum number of shoots and early formation of plantlets using the M medium. Among the treatments, the highest regeneration frequency (87.50 ± 0.20%) and the highest number of PLBs per explant (10.25 ± 0.50) were obtained in P (1.5 g l^? 1)-supplemented cultures, and the plantlets were formed within 18 weeks of culture. BH favoured the development of healthy plantlets, with a maximum fresh weight of 1.02 ± 0.04 g per plantlet.     

Asymbiotic seed germination and in vitro seedling development of the threatened orchid Hoffmannseggella cinnabarina

Hoffmannseggella cinnabarina has not been found in the wild for the last 70?yr in the State of S?o Paulo and, therefore, wild populations of this native orchid are thought to be extinct. This investigation studied seed storage at a low temperature, in vitro germination, and seedling development of H. cinnabarina in order to establish an optimized protocol for propagation, and thus assure species conservation. Seeds of different ages were incubated on Knudson C (KC), Murashige and Skoog, and Vacin and Went media with or without 1???M of N⁶-benzyladenine (BA) and exposed to either 12 or 16?h of light (30???mol?m^?2?s^?1 at 26?±?2°C). Seed surface sterilization was deleterious to 3-mo-old seeds and severely reduced the viability of the 4-mo-old seeds. More mature seeds were not affected by the sterilization procedure. In general, the germination of 4-, 5-, 6-, and 7-mo-old seeds increased when BA was added to the culture medium especially under 16?h of light. Germination rates were highest with 8- and 9-mo-old seeds, and application of BA failed to enhance germination rates further. Developmental studies revealed that this cytokinin reduced seed and protocorm mortality rates; however, protocorm development was negatively affected in its presence. Seedling development was more pronounced when KC medium with 16?h of light was used. Long-term seed storage at 4°C did not provide promising results. The protocol described in this study proved to be efficient and relevant to in vitro seed germination and initial development of H. cinnabarina, and thus will contribute to conservation of this orchid species.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation,micromorphological studies and <Emphasis Type="Italic">ex vitro</Emphasis> rooting of cannon ball tree (<Emphasis Type="Italic">Couroupita guianensis</Emphasis> aubl.): a multipurpose threatened species

In vitro propagation methods using seeds and nodal segments of a 21-year old Couroupita guianensis - a medicinally important but threatened tree have been developed. Hundred percent of the seeds germinated on half strength Murashige and Skoog (MS) medium with 2.0 mg l^?1 indole-3 butyric acid (IBA). Nodal segments were found most suitable for the establishment of cultures. About 90 % explants responded and 4.1 ± 0.23 shoots per node were induced after five weeks of inoculation on MS medium +4.0 mg l^?1 6-benzylaminopurine (BAP). Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoots on fresh medium. Maximum number (8.2 ± 0.17) of shoots were regenerated on MS medium with 1.0 mg l^?1 each of BAP and Kinetin (Kin) + 0.5 mg l^?1 α-naphthalene acetic acid (NAA) with additives (50 mg l^?1 of ascorbic acid and 25 mg l^?1 each of adenine sulphate, L-arginine and citric acid). The multiplied shoots rooted (4.3 ± 0.26 roots/shoot) on half strength MS medium with 2.5 mg l^?1 IBA. All the shoots were rooted ex vitro when pulse treated with 400 mg l^?1 of IBA for five min with an average of 7.3 ± 0.23 roots per shoot. Nearly 86 % of these plantlets were acclimatized within 7–8 weeks and successfully transferred in the field. Biologically significant developmental changes were observed during acclimation particularly in leaf micromorphology in terms of changes in stomata, veins and vein-islets, and trichomes. This study helps in understanding the response by the plants towards outer environmental conditions during acclimatization. This is the first report on micropropagation of C. guianensis, which could be used for the large-scale multiplication, restoration and conservation of germplasm of this threatened and medicinally important tree.     

La fioritura di „Tulipa silvestris“ L. in rapporto alla nutrizione azotata

Many members of the Orchidaceae, the largest vascular plant family in Ecuador, are at risk of extinction. It was therefore considered important to establish an efficient way of clonal propagation based on somatic embryogenesis of Cattleya maxima, a native Ecuadorian orchid. To this end, we evaluated the effect on somatic embryo induction of 12 combinations of 2,4-dichlorophenoxyacetic acid and 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea, as well as three kinds of stresses. Protocorms produced 42% of embryogenic calli on 1/2 Murashige and Skoog (1/2 MS) medium, compared to 96.3% when protocorms were stressed for 6 h with 0.3 M NaCl, followed by cultivation on 1/2 MS medium supplemented with 0.1 mg L^? 1 2,4-D. Our data demonstrated that the combination of either salt (0.3 M NaCl) or osmotic stress (0.4 M sorbitol) with subculture on 2,4-D (0.1 mg L^–1) medium significantly increases the percentage of protocorms with embryogenic callus. The number of embryos per embryogenic callus was not significantly different from that obtained after subculture in growth factor-free medium.     

The novel ethylene antagonist, 3-cyclopropyl-1-enyl-propanoic acid sodium salt (CPAS), increases grain yield in wheat by delaying leaf senescence

Orchid is a major floral crop around the world and Dendrobium hybrids are considered to be one of the most popular orchids. In vitro germination of hybrid seeds is a common practice among orchid growers, however, in many cross pollinations the embryos may not develop to maturity, leading to poor seed germination. The effect of seed maturity and sucrose concentration were investigated via asymbiotic germination of nobile Dendrobium hybrids. Capsules were harvested from two hybrids (Den. Lucky Girl × Den. Second Love ‘Kirameki’ and Den. Lucky Girl × Den. Hamana Lake ‘Kumi’) and one selfing of Den. Second Love ‘Kirameki’ at 2, 3, 4, and 5 months after pollination and immature seeds were taken. Immature seeds from 3- to 5-month old capsules could be successfully germinated on Hyponex based medium. Immature seeds from 4-month old capsules showed greatest germination rate of tested treatments, whereas 3-month old immature seeds showed the least germination. After 6 weeks of in vitro culture, protocorms derived from embryos developed on every concentration of sucrose, but germination was greater at lower concentrations. Greater concentration of sucrose decreased normal-developed protocorms.     

In vitro propagation of <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> Reichenbach fil. through direct adventitious shoot regeneration

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and thidiazuron (TDZ) on direct rhizome induction and shoot formation from rhizome explants of Cymbidium goeringii was explored. Rhizome segments obtained from in vitro seed cultures of C. goeringii were placed on Murashige and Skoog (MS) medium incorporated with 5, 10, 20, or 40 µM 2,4-D and 1, 2, 4, or 8 µM BA or TDZ alone or in combination with 20 µM 2,4-D. The explants developed only rhizomes on MS medium with or without 2,4-D. The highest percent of rhizome formation (100%) was obtained on MS medium incorporated with 20 μM of 2,4-D. The morphology and number of rhizomes varied with the level of 2,4-D in the medium. Direct adventitious shoot formation was achieved on medium incorporated with BA or TDZ. The adventitious shoots produced per explant significantly increased with the supplementation of 2,4-D to cytokinin-containing medium. The highest mean of 21.8 ± 1.8 shoot buds per rhizome segment was obtained in medium fortified with 20 μM 2,4-D and 2 μM TDZ. The greatest percent of root induction (100%) and the mean of 5.3 ± 1.1 roots per shoot were achieved on ½ MS medium incorporated with 2 μM of α-naphthaleneacetic acid. About 97% of the in vitro-produced plantlets acclimatized in the greenhouse. An efficient in vitro propagation protocol was thus developed for C. goeringii using rhizome explants.     

Rapid Multiplication and Induction of Early In Vitro Flowering in Dendrobium Primulinum Lindl

Dendrobium primulinum is an important epiphytic orchid. A successful protocol for mass multiplication and early in vitro flowering was developed. Immature embryos of 4 week after pollination exhibited about 96% germination within 30 days of culture on MS medium containing sucrose (3%) (w/v), NAA and BA (6 and 9 μM) in combination. Protocorm-like bodies (PLBs) formed from the germinating seeds on the germination medium. Rooted plantlets were formed within 2-3 wk on MS medium containing sucrose (3%), NAA and BA (3 and 12 μM in combination) where about 29 shoot/buds produced per cycle of 4 wk interval. The well rooted plantlets produced 4-5 floral buds per spike when they were maintained on MS medium containing sucrose (3%), fresh apple juice (10%) (v/v) for four wk followed by on MS medium freed of apple juice but enriched with NAA and BA (3 and 12 μM respectively). The hardened plantlets were transferred to community potting mix where the about 80% transplants survived after two months of transfer.