Effects of light quality on growth and development of protocorm-like bodies of <Emphasis Type="Italic">Dendrobium officinale</Emphasis> in vitro

The effect of light quality on protocorm-like bodies (PLBs) of Dendrobium officinale was investigated. PLBs of D. officinale were incubated under a number of different light conditions in vitro, namely: dark conditions; fluorescent white light (Fw); red light-emitting diodes (LEDs); blue LEDs; half red plus half blue [RB (1:1)] LEDs; 67% red plus 33% blue [RB (2:1)] LEDs; and 33% red plus 67% blue [RB (1:2)] LEDs. Growth parameters, number of shoots produced per PLB, chlorophyll concentration and carotenoid concentration were measured after 90 days culture. The percentage of PLBs producing shoots was 85% under blue LEDs. In contrast, the percentage of PLBs producing shoots was less than 60% under dark conditions, fluorescent white light and red LEDs. The number of shoots produced per PLB was more than 1.5 times greater under blue LEDs, RB (1:1) LEDs and RB (1:2) LEDs than those cultured under other light treatments [dark, Fw, red LEDs and RB (2:1)]. Chlorophyll and carotenoid concentrations were significantly higher under blue LEDs and different red plus blue LED ratios, compared to other light treatments (dark, Fw and red LEDs). Blue LEDs, Fw, and RB (1:2) LEDs produced higher dry matter accumulations of PLBs and shoots. This study suggests that blue LEDs or RB (1:2) LEDs could significantly promote the production of shoots by protocorm-like bodies of D. officinale and increase the dry matter of PLBs and the accumulation of shoot dry matter in vitro.     

Effects of different spectral lights on <Emphasis Type="Italic">Oncidium</Emphasis> PLBs induction,proliferation, and plant regeneration

The effects of different spectral light distribution on in vitro induction and proliferation of Oncidium protocorm-like bodies (PLBs) and subsequent growth of plantlets were investigated. Shoot tips (5 mm in length) of proliferating shoots of Oncidium “Gower Ramsey” were vertically incubated on 1/2 Murashige and Skoog (MS) medium supplemented with 1.0 mg l⁻¹ 6-benzyladenine (BA), and grown under either monochromatic red light-emitting diodes (LEDs) (RR), blue LEDs (BB), yellow LEDs (YY) or green LEDs (GG). Cultures grown under fluorescent lamps (FL) were used as control. Selected FL-induced PLBs were cut into 3- to 4-mm sections and incubated on MS medium supplemented with 1.0 mg l⁻¹ BA and 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), and grown under RR, BB, YY, GG, or FL. Moreover, FL-differented shoots (15 mm in length with two leaves) were incubated on 1/2 MS medium with 0.5 mg l⁻¹ NAA, and grown under either FL, RR, 10% blue + 90% red LEDs (1BR), 20% blue + 80% red LEDs (2BR), 30% blue + 70% red LEDs (3BR), BB, 80% red + 10% blue + 10% far-red LEDs (RBFr), or 80% red + 10% blue + 10% green LEDs (RBG). Overall, the red light spectrum enhanced induction, proliferation, and the carbohydrate contents of PLBs, as well as subsequent plantlet lengths, while the blue spectrum promoted differentiation, protein accumulation, and enzyme activities in PLBs, as well as pigment content accumulation in PLBs and developing plantlets. The combination of red and blue LEDs resulted in higher energy efficiency as well as dry weight and enzyme activities in these plantlets.     

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.     

Shoot development and plant regeneration from protocorm-like bodies of <Emphasis Type="Italic">Zygopetalum mackayi</Emphasis>

Stem nodal segments of a sympodial orchid, Zygopetalum mackayi, were used as explants to induce protocorm-like body (PLB) formation on a hormone-free 1/2 Murashige and Skoog (1962) modified medium (1/2MS-0) or 1/2MS supplemented with 0.045–4.54 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ] in light. After 1 mo of culture, pale to dark green, compact and irregular nodulars of PLBs formed from the explants. For PLB induction, TDZ had no significant effect on the percentage of PLB formation but promoted mean numbers of PLBs per responding explant at 0.045–4.54 μM. For plant conversion, PLBs were transferred onto the same basal medium devoid of TDZ. After 2–3 mo of culture, these PLBs successfully formed shoots and then roots with normal morphology. For PLB proliferation, TDZ has no significant effects on the fresh weight of PLB aggregates, but there is significantly retarded shoot development at 0.45–4.54 μM after 1 mo of culture. When transferring these PLB aggregates onto hormone-free medium for plant conversion, PLBs derived from TDZ-containing medium showed a decrease of shoot length (0.86–2.08 cm in shoot length) compared to those derived from 1/2MS-0 (2.74 cm in shoot length) after 1 mo of culture. Gibberellin A₃ [GA₃] at 0.29–8.66 μM significantly retarded PLB proliferation, but at 0.03 and 0.29 μM resulted in longer shoot length than the control treatment. Histological studies reveal that shoot development originated from the outer region of PLB aggregates. The young shoots initially connected to each other at their basal tissues with the parental PLBs. Plants were successfully obtained from PLBs and then gradually became more loosely connected with each other as well as with the parental aggregates. Several dozen plants were acclimatized in the greenhouse and showed normal morphology.     

光质及种子大小对普洱地区14种植物种子萌发的影响

以普洱地区14种常见植物种子为材料,在实验室条件下研究了其在白光、黑暗、红光和蓝光条件下的萌发特性,并分析了种子大小与萌发率、萌发速率、萌发开始时间的关系。结果表明:光质对四方蒿、沙针、尖子木、藿香蓟种子萌发率和萌发速率均有显著影响(P0.05)。光质对大叶斑鸠菊、云南山枇花、臭灵丹、车桑子、光萼猪屎豆、葫芦茶、云南地桃花、西南宿苞豆、岗柃、中国宿苞豆10个物种的种子萌发率和萌发速率均没有显著影响(P0.05),以上物种中除中国宿苞豆外,其他物种种子萌发率均在20%以下,处于休眠状态。四方蒿种子在白光(89.9%)和红光(84.7%)下萌发率最高,红光下种子萌发最快(4.93),蓝光下种子萌发开始时间最晚(11.3 d);沙针种子在白光下萌发率最高(80.4%)、萌发速率最快(2.71),在黑暗和蓝光下萌发率较低(43.9%和38%)、萌发速率最慢(0.73和0.85),白光、红光下萌发开始最早(11 d),黑暗条件下萌发开始最晚(21.7 d);尖子木种子萌发率在白光、黑暗、蓝光下均在86%以上,而红光下仅32%且萌发速率最慢(1.29),在蓝光下萌发开始时间最晚(13 d);藿香蓟种子萌发率和萌发速率在红光下最高(分别为71.3%和6.46),黑暗条件下最低(分别为42.5%和2.62);大叶斑鸠菊萌发开始时间在黑暗条件下最早(6 d),其次是白光下(7 d),蓝光和红光下较晚,分别为8 d和7.7 d。14个物种种子的萌发率与种子大小间均有显著负相关关系;种子萌发速率、萌发开始时间与种子大小间也有负相关关系,但不显著;种子大小与萌发率、萌发速率和萌发开始时间的关系不会随着光质的变化而发生变化。     

Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of <Emphasis Type="Italic">Oncidium flexuosum</Emphasis> Sims (Orchidaceae)

The present study describes the direct regeneration of protocorm-like bodies (PLBs) in leaf explants of the tropical species Oncidium flexuosum. The explants were inoculated in a solid, modified Murashige and Skoog (MS) medium with different concentrations of the growth regulator thidiazuron (TDZ) and with or without 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA), and kept away from light or in a 16-h photoperiod. The presence of auxins, 2,4-D, and NAA inhibited the formation of PLBs. The highest frequency of explants that regenerated PLBs (80%) was obtained when they were maintained in a culture medium containing 1.5 μM TDZ under dark conditions. In the same culture medium but under a 16-h photoperiod, 95% of the leaf explants presented necrosis. Therefore, darkness was crucial for the regeneration of PLBs in O. flexuosum leaf explants, which is in disagreement with the literature. PLBs developed from the division of epidermal and subepidermal cells mainly on the adaxial side of the apex region of the explant. Plants with well-developed leaves and roots grew after the PLBs were transferred to growth regulator-free medium under a 16-h photoperiod.     

FACTORS AFFECTING DORMANCY,GERMINATION, AND SEEDLING DEVELOPMENT OF AEGINETIA INDICA L. (OROBANCHACEAE)

Dormancy in seeds of the parasitic phanerogam Aeginetia indica L. can be broken by chemical treatment with sodium hypochlorite, which also helps to control contaminating microflora. A germination medium was developed that suppressed microbial contamination and permitted long-term observation of these slow-germinating seeds. The medium consisted of 10 ppm streptomycin, 10 ppm penicillin, and 10⁻⁵ m indole-3-acetic acid (IAA) (or other growth regulator) in 1 % water agar. Optimum germination range was 25–30 C. Dormancy could also be broken by exposure on agar for several days at 3-5 C (stratification), or by brief exposures (15 min) to 50 C. Continuous light as low as 0.1 ft-c completely inhibited germination on this growth medium. Brief, intermittent light exposures depressed germination. Germination and growth in vitro of nondormant seed of Aeginetina indica L. can be described in five stages: (1) Germination: expansion of spheroidal cells or nodule at micropylar end of the seed, stimulated by growth regulators. Unique stimulation of germination by IAA was noted; (2) Tendril formation: production of a root-hairlike protrusion in response to seedling roots or extract of pea seedlings; (3 and 4) Tendril septation and branching: induced by pea extract; (5) Seedling extension growth: cell growth and division adjacent to nodule by action of various carbohydrate-containing growth factors. This type of growth may bypass tendril formation.     

Evaluation of the effect of three growth regulators in the germination of <Emphasis Type="Italic">comparettia falcata</Emphasis> seeds under <Emphasis Type="Italic">in vitro</Emphasis> conditions

Summary The effect of 3-indoleacetic acid (IAA), 6-furfurylaminopurine (kinetin), and gibberellic acid (GA₃) on germination of the orchid Comparettia falcata was evaluated in a factorial experiment (4×4×4) with Murashige and Skoog (1962) basal medium. It was established that seeds of this orchid could be maintained under aseptic conditions as long as the necessary nutrients and appropriate concentrations of growth regulators were provided. Of the three growth regulators used, IAA significantly decreased seed germination of Comparettia falcata. There was a synergistic effect in the kinetin:GA₃ combination that produced a positive response in both percentage seed germination and development of seedlings. This study describes a single medium-based protocol able to achieve more than 160000 seedlings within 21 wk, starting from a single capsule, sufficient for both large-scale propagation and in vitro conservation of this threatened orchid.     

Direct and callus-mediated protocorm-like body induction from shoot-tips of <Emphasis Type="Italic">Dendrobium chrysotoxum</Emphasis> Lindl. (Orchidaceae)

An efficient method of mass propagation of Dendrobium chrysotoxum Lindl. was developed using a shoot-tip culture system. Both direct and callus-mediated formation of protocorm-like bodies (PLBs) occurred from the basal cut surface of explants. Frequency of callusing was best in the presence of 2 μM thidiazuron (TDZ) or N⁶-benzylaminopurine (BAP). The callus exhibited complete hormone autonomy for growth and differentiation of PLBs and was maintained for 18 months without any exogenous growth regulators, an aspect important for minimising somaclonal variation. However, the rate of callus growth and PLB formation varied with application of cytokinin and auxin. In addition, the callus exhibited a differential sensitivity to the exogenous cytokinins. While BAP promoted callus growth and PLB differentiation, TDZ was inhibitory to callus mediated PLB formation and caused extensive necrosis of callus. Although α-naphthaleneacetic acid (NAA) had no significant effect on the induction of callus, subsequent growth was best in its presence. Using a 3-month subculture period, a 69-fold increase in callus weight was achieved with 0.5 μM NAA, while as many as 133 PLBs could be obtained per 100 mg callus in the presence of 1 μM NAA. For direct PLB formation, the optimum cytokinin dosage was dependent upon the type of cytokinin used. While TDZ was most effective at a concentration of 1 μM (15 PLBs per explant), for similar PLB yield the application of 8 μM BAP was essential.     

In situ seed baiting techniques in <Emphasis Type="Italic">Dendrobium officinale</Emphasis> Kimuraet Migo and <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl. : the endangered Chinese endemic <Emphasis Type="Italic">Dendrobium</Emphasis> (Orchidaceae)

The new in situ seed baiting method using seed packets to assess germination of orchid species within soil provides a means of locating, collecting, and identifying specific fungi that are involved in the lifecycle of orchids in the wild and investigating their relationship with the orchids under natural field conditions. Two isolates (SHH44, SHH53) originating from the seedlings found in the seeds packets in situ were demonstrated to support seed germination and seedling development (in vitro) of two endangered Chinese endemic herbs, Dendrobium officinale and Dendrobium nobile (Orchidaceae). Advanced protocorm development (Stage 3 and greater) in this study of these species of Dendrobium only occurred under the 12/12 h L/D photo-period indicated that illumination may play an important role in seedling recruitment of terrestrial orchid species in their natural environment. The information provided in this study may prove invaluable in the conservation of D. officinale and D. nobile and congener species in China, especially given their rare status.     

Flowering of Cultivated Green and SAN 9789-Treated Chenopodium rubrum Plants Exposed to White,Blue, and Red Light

Green plants and plants devoid of photosynthetic pigments were compared with regard to their ability to flower under various growth conditions. Green plants of Chenopodium rubrum L. and plants treated with norflurazon SANDOZ-9789 (SAN) were grown on sucrose-containing media with or without hormones (GA₃, BA, IAA, ABA) under short-day photoperiodic or continuous illumination with white, blue, or red light. Green and SAN-treated albino plants produced flowers only under short-day conditions. The flowering of green plants was independent of the presence of sucrose and hormones in the medium as well as of the light quality. The albino plants produced flowers under white and blue light but did not flower in red light. The addition of GA₃ or BA to the medium induced flowering of albino plants exposed to red light. The functional interaction of photoreceptors in the flowering control is discussed.     

In vitro morphogenetic responses from obligatory apomictic Taraxacum belorussicum Val. N. Tikhom seedlings explants

Taraxacum belorussicum Val. N. Tikhom, a poorly known and obligatory apomictic species, is an attractive plant material for studying the embryological, genetic and molecular mechanisms of apomixis. This work aims to obtain an efficient protocol for Taraxacum belorussicum regeneration. Four types of explants (cotyledons, hypocotyls, meristems and roots) that were taken from 2-weeks-old seedlings were used for in vitro cultures, and a fast and efficient protocol of T. belorussicum regeneration was obtained. Various ½ MS-based media containing IAA (5.71 µM), TDZ (4.54 µM) and PSK (100 nM) were chosen to assess the morphogenetic abilities of selected T. belorussicum explants. Studies on the role of PSK were done in three independent experiments, where the most significant factors were always light and darkness. All explants produced callus by the third day of culture and adventitious shoots after 7 days, although in an asynchronous indirect manner, and with different intensities for all explant types. The most preferred medium culture for hypocotyl, cotyledon and meristem explants was ½ MS?+?TDZ, and ½ MS?+?IAA?+?TDZ?+?PSK for roots which were the only explant sensitive to PSK. A short darkness pretreatment (8 days) in PSK medium was found suitable to enhance organogenesis. Secondary organogenesis was observed for regenerated plants on meristem explants from the ½ MS?+?IAA?+?TDZ?+?PSK medium. A weak somatic embryogenesis was observed for hypocotyl and cotyledon explants from ½ MS?+?IAA?+?TDZ and ½ MS?+?IAA?+?TDZ?+?PSK media. Histological and scanning electron microscope images (SEM) of T. belorussicum confirmed indirect organogenesis and somatic embryogenesis. Plant material treated with aniline blue solution revealed the presence of callose in the cell walls of cotyledon and hypocotyl explants. The presence of extracellular matrix (ECM) and heterogenic structure of callus was also verified by scanning electron microscopy and light microscopy, confirming the high morphogenetic ability of T. belorussicum.

     

Effect of blue light on indoor seedling culture of <Emphasis Type="Italic">Saccharina japonica</Emphasis> (Phaeophyta)

Saccharina japonica is a brown alga that has been commercially cultured on a large scale in China. Integrating the light condition under seawater and the adaptation of Saccharina to this condition, it is expected that blue light would be beneficial to Saccharina culture system. Consequently, the detailed effect of blue light on the key stages during indoor seedling culture of S. japonica was investigated in this study. Irradiances and light qualities had little effect on zoospore attachment and germination. Egg formation occurred sooner under blue light than white light. Under optimum irradiances, 95 ± 4% female gametophytes gave rise to eggs in 6 d under blue light, while it took 12 d for over 90% formation of eggs under white light. Over a culture period of 3 weeks, mean sporeling length and width under blue light was 1.39 and 1.56 times of that under white light, respectively, while the mean sporeling size obtained under red light was only 25% of that under white light. The higher growth rate under blue light was largely due to higher photosynthetic efficiency, as indicated by chlorophyll fluorescence of photosystem II. In addition, the mean ratio of sporeling width to length under blue light was significantly higher than that under white light. These results suggest that blue light would be superior to white light for indoor seedling culture of S. japonica. Based on these findings, an improved S. japonica seedling culture system is discussed.     

Asymbiotic seed germination and in vitro seedling development of Habenaria macroceratitis (Orchidaceae), a rare Florida terrestrial orchid

Continuing loss of native orchid habitat has lead to an increased emphasis on orchid conservation. Major obstacles in the production of native orchid seedlings for use in conservation have been: (1) development of efficient and reliable seed germination protocols and (2) an understanding of early seedling growth and development. Effects of six asymbiotic media (Modified Lucke, Murashige & Skoog, Lindemann, Vacin & Went, Malmgren Modified, Knudson C), four exogenous cytokinins (BA, Zea, Kin, 2-iP), and three photoperiods (0/24, 16/8, 24/0 h L/D) were examined on seed germination and early protocorm development of Habenaria macroceratitis, a rare native Florida terrestrial orchid. Finally, the effects of three photoperiods (8/16, 12/12, 16/8 h L/D) on in vitro seedling development were examined. Percent seed germination was highest on both LM and KC after seven weeks culture (LM = 89.1%, KC = 89.2%); however, protocorm development was enhanced on MM after both seven and 16 weeks. Both zeatin and kinetin at 1 μM enhanced seed germination (Zea = 58.1%, Kin = 47.2%). Final percent seed germination (91.7%) and protocorm development (Stage 4) was increased in the absence of light (0/24 h L/D). In␣vitro seedlings cultured under 8/16 h L/D conditions produced the highest number of tubers per seedling (1.06) with the greatest tuber (42.7 μg) and shoot (fwt = 69.5 μg) biomass and tuber diameter (3.1 mm).     

Adventitious shoot induction and somatic embryogenesis with intact seedlings of several hybrid seed geranium (Pelargonium x hortorum Bailey) varieties

Summary Intact seedlings of hybrid seed geranium (Pelargonium x hortorum Bailey) were tested for their ability to produce adventitious shoots and somatic embryos by direct culture of mature seeds on Murashige and Skoog (1962) medium (MS) supplemented with growth regulators BAP, BAP + IAA, or thidiazuron (TDZ). Ten varieties were tested in the presence of different BAP concentrations, four with BAP + IAA, and two with TDZ. Varieties used in this study differed in their response to BAP in the medium. Multiple adventitious shoots were produced by seven of the ten varieties tested. Multiple adventitious shoots were induced at all levels of TDZ in the medium. TDZ also induced callusing from roots and direct embryogenesis from intact hypocotyls. Adventitious shoots were separated, rooted and transferred to soil where they grew as normal healthy plants and flowered.Abbreviations BAP N⁶-benzylaminopurine - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) - TDZ thidiazuron     

In vitro germination,protocorm formation and plantlet development of mature versus immature seeds from several <Emphasis Type="Italic">Ophrys</Emphasis> species (Orchidaceae)

We investigated the effect of genotype, seed maturity and culture medium on the in vitro germination and development of protocorms and plantlets from seeds of 13 different Ophrys species (O. apifera, O. attica, O. cornuta, O. delfinensis, O. ferrum-equinum, O. lutea, O. mammosa, O. speculum, O. spruneri, O. umbilicata, O. argolica, O. irricolor and O. tenthredinifera) collected in Greece, some of which are endemic to this country. Mature seeds (10 months after collection) and immature seeds (2 months after anthesis) were cultured in a coconut milk-enriched or a pineapple-enriched medium (CEM or PEM, respectively). The highest percentage of callogenesis (96%) was observed in immature seeds of O. delphinensis in the CEM, while the highest percentage of protocorm formation (52%) was observed in mature seeds of O. spuneri in the CEM. Protocorm formation was significantly lower in immature seeds than in mature seeds in both culture media. Eventually almost all of the transferred protocorms developed to plantlets, which later formed minitubers. PEM appeared to be the most suitable for the development of minitubers from plantlets. All of the factors investigated—as well as their interactions—significantly affected callogenesis and protocorm formation. The results are discussed with the perspective of applying an improved protocol for in vitro seed germination and plantlet formation in several under-utilized Ophrys species.Abbreviations CEM Coconut milk-enriched medium - CLT Callus-like tissue - PEM Pineapple-enriched medium - PPFD Photosynthetic photon flux density     

In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid,Dendrobium aphyllum (Orchidaceae)

Orchid conservation efforts, using seeds and species-specific fungi that support seed germination, require the isolation, identification, and germination enhancement testing of symbiotic fungi. However, few studies have focused on developing such techniques for the epiphytes that constitute the majority of orchids. In this study, conducted in Xishuangbanna Tropical Botanical Garden, Yunnan, China, we used seeds of Dendrobium aphyllum, a locally endangered and medicinally valuable epiphytic orchid, to attract germination promoting fungi. Of the two fungi isolated from seed baiting, Tulasnella spp. and Trichoderma spp., Tulasnella, enhanced seed germination by 13.6 %, protocorm formation by 85.7 %, and seedling development by 45.2 % (all P?Epulorhiza, another seed germination promoting fungi isolated from Cymbidium mannii, also enhanced seed germination (6.5 %; P?P?Trichoderma suppressed seed germination by 26.4 % (P?Tulasnella was the only treatment that produced seedlings. Light increased seed imbibition, protocorm formation, and two-leaved seed development of Tulasnella inoculated seeds (P?Tulasnella be introduced for facilitating D. aphyllum seed germination at the protocorm formation stage and that light be provided for increasing germination as well as further seedling development. Our findings suggest that in situ seed baiting can be used to isolate seed germination-enhancing fungi for the development of seedling production for conservation and reintroduction efforts of epiphytic orchids such as D. aphyllum.     

Growth regulator-induced betacyanin accumulation and dopa-4,5-dioxygenase (<Emphasis Type="Italic">DODA</Emphasis>) gene expression in euhalophyte <Emphasis Type="Italic">Suaeda salsa</Emphasis> calli

Suaeda salsa calluses cultured in darkness for 28 d were transferred to Murashige and Skoog (MS) media containing various growth regulators under white light conditions for 10 d to investigate cell growth, betacyanin accumulation, and expression of dopa-4,5-dioxygenase (DODA). Callus growth was markedly promoted when 0.2 mg·L⁻¹ 2,4-D and 0.5 mg·L⁻¹ 6-BA were added to the MS medium. Surprisingly, of the auxins tested, IAA had no effect on betacyanin content, but 2,4-D strongly decreased betacyanin content. Betacyanin content was positively correlated with 6-BA concentrations in the range of 0.1–2.0 mg·L⁻¹. DODA mRNA levels were consistent with the response of betacyanin content to exogenous growth regulators. These results suggest that betacyanin metabolism in S. salsa calluses is regulated under white light conditions by growth regulators through the regulation of genes such as DODA that are involved in betacyanin synthesis.     

High-efficiency cryopreservation of the medicinal orchid <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl.

An efficient protocol for cryopreservation of protocorm like bodies (PLBs) of Dendrobium nobile, based on encapsulation–dehydration (ED) and encapsulation–vitrification (EV), was established. In both cryogenic procedures, PLBs were initially osmoprotected with a mixture of 0.4 M sucrose and 2 M glycerol, incorporated in the encapsulation matrix [comprising 3% (w/v) sodium alginate and 0.1 M CaCl₂]. Out of the two methods, EV resulted in higher survival (78.1%) and regrowth (75.9%) than ED (53.3 and 50.2% respectively). Incorporation of 0.4 M sucrose and 2 M glycerol in the encapsulation matrix resulted in higher survival percentage after cryopreservation. In both the cases (ED and EV), shoots regenerated from cryopreserved PLBs with an intermediary PLB formation. Regenerated shoots were successfully rooted in the medium containing 1.5 mg/l Indole-3 butyric acid. Successful acclimatization of plantlets was obtained in the compost containing brick pieces and charcoal chunks (1:1) + a top layer of moss with a maximum survivability (82%). EV method proved to be most appropriate way to cryopreserve the PLBs of D. nobile. Regenerated plantlets showed normal morphology as that of control plants.     

In vitro germination and seedling development of two European orchid species,Himantoglossum jankae Somlyay,Kreutz & Óvári and Spiranthes spiralis (L.) Chevall.

New protocols for successful asymbiotic seed germination of two European orchid species, Himantoglossum jankae Somlyay, Kreutz & Óvári and Spiranthes spiralis (L.) Chevall., were established in this study. The influence of two basal media, organic supplements, illumination, and cytokinins on germination, protocorm formation, and seedling development was examined. A strong species-specific dependence on illumination conditions and nutrient medium composition was observed. Improved germination under continual darkness indicated that H. jankae had negatively photoblastic seeds, while S. spiralis seeds could germinate under both light conditions examined in this study, but slightly faster development was achieved under the 16-h photoperiod protocol. The H. jankae seeds cultured on Knudson C medium failed to germinate in any medium combination, irrespective of the organic supplement use. The highest germination rate of H. jankae was achieved on Malmgren medium supplemented with coconut water, peptone, and L-glutamine, indicating that germination of this species was amino acid–dependent. Although S. spiralis seeds germinated on all medium combinations, Knudson C basal medium (without supplements) yielded better results when compared with the Malmgren medium. The best S. spiralis seedling development was achieved on the medium supplemented with 6-(γ,γ-dimethylallylamino) purine, while the cytokinin 6-benzyladenine promoted H. jankae seedling development. With the implementation of these protocols, well-developed seedlings were acclimatized to greenhouse conditions after 7 mo in culture.