Tissue culture independent transformation for Corchorus olitorius

An efficient microprogation protocol has been developed for Dendrobium densiflorum Lindl. ex Wall., a traditional medicinal plant, through protocorm-like bodies (PLBs) from nodal stem segments using 6-benzylamino-purine (BAP) and the lanthanoid neodymium. The highest percentage of explants producing PLBs (72%), with an average of 15 PLBs per explant, was induced by culturing stem segments on Murashige and Skoog (MS) medium supplemented with 5.0 mg l⁻¹ BAP. The newly formed PLBs proliferated well on the basal MS medium and completely converted into shoots on MS medium containing 2.0 mg l⁻¹ BAP. Shoots produced an average of 22 roots per plantlet when cultured on MS medium supplemented with 2.0 mg l⁻¹ neodymium nitrate. Healthy plantlets with well-developed roots were successfully acclimatized. The obtained result suggests that the lanthanoids can be used to effectively initiate rooting in the micropropagation and conservation of D. densiflorum.     

Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination

A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both -glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3–1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l^–1 hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.     

石斛兰转ACS反义基因抗性原球茎及转化植株的筛选与鉴定

该试验就石斛兰转化ACS(1-氨基环丙烷-1-羧酸合成酶)反义基因的不同筛选方法和筛选处理对抗性原球茎筛选的影响,以及石斛兰转基因植株的再生与鉴定进行研究.结果表明:(1)石斛兰原球茎经带有gus报告基因和ACS反义基因的农杆菌LBA4404侵染共培养5d后除菌,采用逐渐提高选择压浓度的延迟筛选,并在低选择压浓度下切割而高选择压浓度下不切割的处理方式为抗性原球茎的最佳筛选途径,抗性原球茎获得率可达14.97％.(2)抗性原球茎繁殖时应逐渐降低选择压浓度,且在低选择压浓度下进行切割处理,繁殖倍数达到1.15倍,且原球茎生长势好.(3)抗性原球茎在1/2 MS+0.5 mg/L 6-BA培养基中的分化率达到73.85％;107株无根小苗培养于1/2 MS+1.0 mg/L NAA+50.0 mg/L Km(卡那霉素)+100.0 mg/L Cef(头孢霉素)培养基中进行生根培养,共获得了13株具有卡那霉素抗性的转化植株,转化效率达到12.15％.(4)转化植株经报告基因产物GUS组织化学检测和gus的PCR检测,证实带ACS反义基因的T-DNA已整合进石斛兰基因组中,且转基因植株在形态上与未转基因植株无明显差别,3株转基因植株移栽2个月后均已成活.     

Efficient production of protocorm-like bodies and plant regeneration from flower stalk explants of the sympodial orchid <Emphasis Type="Italic">Epidendrum radicans</Emphasis>

Summary A simple and efficient micropropagation method was established for direct protocorm-like body (PLB) formation and plant regeneration from flower stalk internodes of a sympodial orchid, Epidendrum radicans. Small transparent tissues formed on surfaces and cut ends of flower stalk internodes on a modified half-strength Murashige and Skoog basal medium with or without thidiazuron (TDZ) after 1–2 wk of culture. In the light, the transparent tissues enlarged and turned into organized calluses on most of the explants. However, PLBs formed only on a medium supplemened with 0.45 μM TDZ within 2 mo. of culture. Sucrose, NH₄NO₃, and KNO₃ were used in media to test their effects on PLB proliferation and shooting. The best response on number of PLBs per tube was 23.6 at 40 gl⁻¹ sucrose, 825 mgl⁻¹ NH₄NO₃, and 950 mgl⁻¹ KNO₃, and the highest number of PLBs with shoots was found at 10 gl⁻¹ sucrose, 825 mgl⁻¹ NH₄NO₃, and 950 mgl⁻¹ KNO₃. Homogenized PLB tissues produced by blending were used to test the effects of four cytokinins [TDZ, N⁶-benzyladenine (BA), zeatin-riboside, and kinetin] on PLB proliferation and shoot formation. The best responses on number of PLBs per tube, proliferation rate, and number of PLBs with shoots per tube were obtained at 4.44 μM BA, 0.28 μM zeatin-riboside, and 1.39 μM kinetin, respectively. Normal plantlets converted from PLBs on the same TDZ-containing medium after 1 mo. of culture. The optimized procedure required about 12–13 wk from the initiation of PLBs to plantlet formation. The regenerated plants grew well with an almost 100% survival rate when acclimatized in a greenhouse.     

Micropropagation of Aerides maculosum lindl. (Orchidaceae)

Summary Young leaf segments from plants growing both in vivo and in vitro were cultured on Murashige and Skoog (MS) medium supplemented with auxins [naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D)], cytokinins [kinetin (KN) and N⁶-benzyladenine (BA)] and coconut liquid endosperm (CW). The explants from mature leaves did not show any growth and turned necrotic, while those obtained from juvenile leaves growing in vitro developed protocorm-like bodies (PLBs) at their cut surfaces within 4–8 wk depending on the growth medium. An optimum of 18 PLBs developed from leaf explants on medium supplemented with 2.0 mg l⁻¹ (8.87 μM) BA. Upon subculture in basal MS medium, the PLBs differentiated into plantlets within 6–8 wk. The resulting plantlets were successfully transferred to vermiculite initially and subsequently to potting mixture; 84% of the plantlets survived after 3 mo. of transplantation.     

Plant regeneration via protocorm-like body formation and shoot multiplication from seed-derived callus of a maudiae type slipper orchid

Tiny seeds from 5-month-old green capsules of a maudiae type slipper orchid, Paphiopedilum Alma Gavaert, were induced to form totipotent callus on 1/2 strength MS medium supplemented with 22.60 μM 2,4-D and 4.54 μM TDZ in darkness. The callus was proliferated more and maintained without any morphogenesis on the same medium with a 2-month interval of subculture for more than 2 years. When transferred to 1/2 MS medium supplemented with 26.85 μM NAA, an average of 4.7 protocorm-like bodies (PLBs)/shoot buds formed from each explant after 120 days of culture. After another 72 and 240 days of culture on the same medium, 25 shoot buds and eventually 75 plantlets were obtained through shoot multiplication from the original culture. Kinetin at 4.65 μM was suitable for shoot multiplication and could induce an average of 3.0 shoots from a single young shoot after 60 days of culture. The regenerated plantlets grew normally when transplanted to containers with sphagnum moss in a shaded greenhouse.     

In vitro culture of Doritaenopsis: comparison between formation of the hyperhydric protocorm-like-body (PLB) and the normal PLB

Characteristics of the hyperhydric protocormlike-bodies (hPLBs) and the normal PLBs (nPLBs) of Doritaenopsis are morphologically and ontogenetically compared. The hPLBs have a translucent and turgid appearance which is due to a lack of air volume in the intercellular spaces. The hPLBs have a lower capacity of shoot formation but a higher capacity of differentiation of new PLBs than the nPLBs. The new PLBs derived from the hPLBs can be recovered from hyperhydricity using a medium containing potato juice. This suggests the possibility that hPLBs can be used for the in vitro propagation of orchid plants.Abbreviations PLB Protocorm-Like-Body - mVW Modified Vacin and Went Medium     

Ploidy mosaics: does endopolyploidy in explants affect the cytogenetic stability of orchids regenerated from PLBs?

The induction and regeneration of protocorm-like bodies (PLBs) is a morphogenetic pathway widely used for orchid micropropagation. As endopolyploidy, i.e., the coexistence of cells with different ploidy levels, is a common feature in orchid tissues, a natural question arises when using somatic tissues as explants for orchid micropropagation: does endopolyploidy in explants affect the cytogenetic stability of regenerated plantlets? To answer this question, Epidendrum fulgens was used as a model plant, and flow cytometry was used to analyze endopolyploidy in pollinia, petals, labella, leaf bases, leaf tips, root tips, and protocorm bases and apices, which were subsequently used as explants for PLB induction and plant regeneration. Ploidy screenings showed contrasting ploidy patterns in samples, endopolyploidy being detected in all tissues, with C-values ranging from 1 to 16C. Protocorm bases and root tips presented the highest proportion of endopolyploidy, while petals and protocorm apices showed the lowest proportion. Flower parts exhibited high oxidation for PLB induction and pollinia failed to produce PLB or callus. The highest induction rate occurred at 10 µM TDZ, with 92%, 22%, and 0.92% for protocorm bases, leaves, and root tips, respectively. Plantlets were more easily regenerated from PLBs induced from protocorm bases than from leaves and roots. Doubled ploidy levels were registered in a proportion of 11% and 33% for PLB-regenerated plantlets obtained from protocorm bases and leaf bases, respectively, which was not directly associated with the proportion of endopolyploid cells or cycle value of explants.

     

Relative importance of maltose and sucrose supplied during a 2-step potato microtuberization process

A 2-stage in vitro tuberization process comprising first micropropagation via nodal explants and then tuber induction in the resultant in vitro plantlets was studied using 2 cultivars of potato, Iwa and Daeji. In particular, the effects on both plantlet growth and subsequent in vitro tuberization of Murashige and Skoog (1962) basal medium containing either sucrose or maltose, each at 3 % (w/v), used for micropropagation were investigated. Sucrose and maltose were found to be equally effective in supporting development of vigorous plantlets from the nodal explants of both potato cultivars. Upon transfer to a medium with an optimised level of sucrose (i.e. 8 %, w/v) for in vitro tuberization, only the plantlets previously grown in the sucrose-containing medium were capable of forming more microtubers of the larger size category (greater than 0.5 g). The relative importance of sucrose supply at the mircropropagation stage was further confirmed when the resultant plantlets grown in the 3 % sucrose-containing medium were transferred to an in vitro tuberization medium containing either sucrose or maltose, each at 8 % (w/v). In this experiment, maltose and sucrose had indistingushable effects on in vitro tuberization.     

Ploidy doubling by in vitro culture of excised protocorms or protocorm-like bodies in <Emphasis Type="Italic">Phalaenopsis</Emphasis> species

Endopolyploidy was observed in the protocorms of diploid Phalaenopsis aphrodite subsp. formosana with ploidy doubling achieved by in vitro regeneration of excised protocorms, or protocorm-like bodies (PLBs). Thirty-four per cent of the PLBs regenerated from the first cycle of sectioned protocorms were found to be polyploids with ploidy doubled once or twice as determined by flow-cytometry. The frequency of ploidy doubling increased as the sectioning cycles increased and was highest in diploid followed by the triploid and tetraploid. Regeneration of the endopolyploid cells in the tissue of the protocorms or PLBs is proposed as the source of the development of ploidy doubled plantlets. The frequency of ploidy doubling was similar in seven other Phalaenopsis species, although the rate of increase within cycles was genotype specific. In two species, a comparison of five parameters between 5-month-old diploid and tetraploid potted plants showed only the stomata density differed significantly. The flowers of the tetraploid plant were larger and heavier than those of the diploids. This ploidy doubling method is a simple and effective means to produce large number of polyploid Phalaenopsis species plants as well as their hybrids. The method will be beneficial to orchid breeding programs especially for the interspecific hybridization between varieties having different chromosome sizes and ploidy levels.     

Cryopreservation of <Emphasis Type="Italic">Dendrobium candidum</Emphasis> Wall. ex Lindl. protocorm-like bodies by encapsulation-vitrification

Protocorm-like bodies (PLBs) of Dendrobium candidum Wall. ex Lindl., orchid, were successfully cryopreserved using an encapsulation vitrification method. PLBs were precultured in liquid Murashige and Skoog (MS) medium containing 0.2 mg l⁻¹ α-naphthalene acetic acid and 0.5 mg l⁻¹ 6-benzyladenine enriched with 0.75 M sucrose, and grown under continuous light (36 μmol m⁻² s⁻¹) at 25 ± 1°C for 5 days. PLBs were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dripped in a 0.5 M CaCl₂ solution containing 0.5 M sucrose at 25 ± 1°C and left for 15 min to form Ca-alginate beads (about 4 mm in diameter). Then, these were dehydrated with a plant vitrification solution 2 (PVS₂) consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) dimethyl sulfoxide in 0.5 M sucrose, pH 5.8, for 150 min at 0°C. Encapsulated and dehydrated PLBs were plunged directly into liquid nitrogen for 1 h. Cryopreserved PLBs were then rapidly re-warmed in a water bath at 40°C for 3 min and then washed with MS medium containing 1.2 M sucrose for three times at 10 min intervals. Within 60 days, plantlets with the cryopreserved PLBs developed normal shoots and roots, and without any observed morphological abnormalities, were obtained. The survival rate of encapsulated-vitrified PLBs was above 85%. Thus, this encapsulation-vitrification method was deemed promising for cryopreservation of PLBs of D. candidum.     

Cryopreservation of immature seeds of Bletilla striata by vitrification

An efficient protocol was established for the cryopreservation of immature seeds of a terrestrial orchid, Bletilla striata. Immature seeds collected 2–4 months after pollination (MAP) were treated using three different cryogenic procedures: (1) direct plunging into liquid nitrogen, (2) vitrification, and (3) vitrification with preculture. When immature seeds collected 3 MAP and 4 MAP were precultured for 3 days on New Dogashima medium supplemented with 0.3 M sucrose and cryopreserved by vitrification, the survival rate after preservation, as assessed by staining with 2,3,5-triphenyltetrazolium chloride, was 92% and 81%, respectively. Immature seeds thus treated showed no decrease in germination rate relative to untreated immature seeds, and they developed into normal plantlets in vitro.     

Plant regeneration from stem nodal segments of <Emphasis Type="Italic">Orthosiphon stamineus</Emphasis> benth., a medicinal plant with diuretic activity

Summary A micropropagation method for Orthosiphon stamineus, using stem nodal segments, has been developed. The highest number of regenerated shoots was obtained on Murashige and Skoog (MS) medium supplemented with 6.7 μM benzyladenine with the formation of an average of 6.1 shoots per explant over a period of 4 wk. The number of shoots increased with longer culture duration on proliferation medium. Multiple shoots which were maintained on the proliferation medium for 6 wk had the highest proliferation rate. Separation of multiple shoots and culturing in larger flasks significantly promoted the growth and formation of plantlets. All the in vitro plantlets survived when transferred to the field and showed no significant morphological differences from the mother plants.     

Micropropagation of Spathoglottis plicata

A rapid and reliable micropropagation method was established for Spathoglottis plicata. Nodal and leaf explants dissected from 8-month-old pot-grown seedlings were cultured on charcoal-amended Murashige and Skoog medium supplemented with 16 combinations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA) at concentrations of 0.54–10.74 μm. Regeneration of protocorm-like bodies (PLBs) and subsequent plantlet development were observed from 98.5% of the nodal explants. Only 6.5% of leaf explants and occasionally some root segments (dissected from regenerated plantlets) were able to produce PLBs and then plantlets. The optimum plant growth regulator (PGR) combination for maximal PLB regeneration was 5.37 μm NAA and 0.44 μm BA. The best combination of PGR for plantlet development was 2.69–10.74 μm NAA and 8.88 μm BA. The NAA to BA ratios for maximal PLB induction and plantlet development were 12.2 and 0.3–1.2, respectively. Regenerated PLBs and plantlets, when cut into pieces of less than 1 mm and subcultured onto the above media, regenerated new PLBs and plantlets in another 3 months. Received: 20 February 1997 / Revision received: 27 May 1997 / Accepted: 16 June 1997     

Mass multiplication of protocorm-like bodies using bioreactor system and subsequent plant regeneration in Phalaenopsis

Protocorm-like bodies (PLBs) formed on leaf segmentsin vitro were used as explants for bioreactor cultures. Continuous immersion cultures (air lift column and air lift-balloon bioreactor), and temporary immersion cultures (with or without charcoal filter attached) were used for the culture of PLB sections. A temporary immersion culture with charcoal filter attached was most suitable for PLB culture. About 18,000 PLBs were harvested from 20 g of inoculum (∼1000 PLB sections) in 2 l Hyponex medium after 8 weeks of incubation. Aeration in a bioreactor at 0.5 or 2.0 volume of air per volume of medium min⁻¹ (vvm) yielded similar levels of biomass production. PLBs grown in bioreactors were cultured on solid Murashige and Skoog, Vacin and Went, Knudson C, Lindemann and Hyponex media. Hyponex medium was found to be suitable for conversion of PLBs into plantlets and 83% of PLBs transformed into plantlets on this medium. The feasibility of using PLBs for large-scale micropropagation was evaluated for scaled-up liquid cultures in bioreactors, rate of proliferation, and regeneration. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro micropropagation of Primula scotica: a rare Scottish plant

A simple micropropagation method is reported for Primula scotica, a rare plant, endemic to the North of Scotland. The technique involves the clonal proliferation of seed-derived plantlets on either hormone free tissue culture medium or on medium containing benzyl amino purine and indole acetic acid. Average multiplication rates of 4–6 were obtained for plants grown on plant growth regulator supplemented medium. The micropropagation method was applied to four different clones of P. scotica and clonal differences were observed in relation to media type. Some plants from certain clones did display hyperhydricity, however, this was circumvented by using frequent sub-culturing intervals and transferring the plants to hormone-free medium. Plantlets rooted on both media types and displayed normal, true-to-type rosette morphology. The cultures did not callus and development proceeded via shoot and root production only. The in vitro-grown plants could be transferred to ex vitro conditions and a range of growth substrates were assessed for their efficacy in supporting ex vitro growth, with a view to developing longer-term strategies for the transfer and reintroduction of micropropagated P. scotica plants into natural habitats. The simple method described in this paper may offer the potential of being applied to other endangered Primula spp.     

Agrobacterium tumefaciens-mediated transformation of Oncidium and Odontoglossum orchid species with the ethylene receptor mutant gene etr1-1

Oncidium and Odontoglosum orchid species have reduced display lives and are thus commercially less important than Phalaenopsis. One approach to prolonging display life permanently is to transform Oncidium and Odontoglossum with the ethylene receptor mutant gene etr1-1 from Arabidopsis under control of a flower specific promoter; this should reduce their sensitivity to exogenous ethylene. To achieve this it will be necessary to establish an efficient regeneration protocol using somatic embryogenesis and a routine Agrobacterium tumefaciens-mediated transformation procedure. Protocorm-like bodies (PLBs) of both orchid genera were regenerated from leaf tip explants. Leaf tips and PLBs, cultured in liquid and solid media, were compared as targets for genetic transformation. No transgenic shoots were obtained from leaf tips, while PLBs of Oncidium and Odontoglossum cultured on solid medium were successfully transformed with an expression vector containing nptII and gus genes driven by the cauliflower mosaic virus (CaMV) 35S promoter. Applying the A. tumefaciens strain EHA 105, transformation efficiencies of 1.3–2.7% were achieved for the investigated genotypes. Transformation with etr1-1 gene was achieved subsequently. Oncidium ‘Sweet Sugar’ has been successfully transformed and validated by PCR and Southern analysis.     

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.     

Formation of endomycorrhizas by an achlorophyllous orchid,Erythrorchis ochobiensis, andAuricularia polytricha

To test the mycorrhizal function of heterobasidiomycetous fungi on achlorophyllous orchids and to examine the symbiotic fungal range of a myco-heterotrophic orchid,Erythrorchis ochobiensis, synthetic cultures of the orchid seed were carried out withAuricularia polytricha isolates from Japan and Mexico. After three and a half mo of incubation, 57.0–70.7% of seeds germinated but none of them showed further growth. When cultured on peat moss at 25°C, the germination rate was 8.7% in the presence of Mexican isolate and 18.0% in the presence of Japanese isolate. Some germinated seeds developed into protocorms, and several seeds incubated with the Mexican isolate developed into plantlets after 5 mo. Pelotons were observed in the cells of protocorms and roots. The results indicated that some heterobasidiomycetous fungi could form endomycorrhizas with a myco-heterotrophic orchid. The results also showed that the symbiont ofE. ochobiensis extends, at least experimentally, to Heterobasidiomycetes. The variances of germination rate and seedling growth were suggested to be affected by the difference of isolates and culture conditions.     

In vitro propagation of threatened terrestrial orchid, Malaxis khasiana Soland ex. Swartz through immature seed culture

Rapid in vitro propagation of the terrestrial orchid, M. khasiana through immature seed culture was achieved. Immature seeds of 8-9 week after pollination (WAP) cultured on MS medium (2% sucrose) supplemented with 500 mgl(-1) casein-hydrolysate and 1 microM N6-benzyladenine (BA) exhibited germination of 75% seeds after 107 days of culture and subsequently supported the development of PLBs. Subsequent culture on MS medium enriched with 6 microM of indole-3-acetic acid (IAA), 18 microM each of BA and kinetin induced multiple shoots and plantlets. Transfer of PLBs to MS medium with 0.1% activated charcoal (AC) facilitated rapid proliferation of PLBs, while AC at 0.2% favored shoot bud induction and rhizome enlargement. The plantlets, developed on medium with IAA, BA and kinetin, after hardening in vitro for 8-10 weeks were planted in community pots and transferred to poly-house. The plantlets showed 65% survival under field conditions.