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.     

The role of thin cell layers in regeneration and transformation in orchids

Thin cell layers (TCLs) offer a simple yet effective protocol that has contributed to major advances in clonal micropropagation of orchids. TLCs have been successfully used for protocorm-like body (PLB) and callus induction in Aranda, Coelogyne cristata, Cymbidium spp., Dendrobium spp., Doritaenopsis, Paphiopedilum, Renanthera, Rhynchostylis, Spathoglottis, and Xenikophyton. TCLs have also been a bulwark for genetic transformation studies of select genera. This review takes an in-depth look at how TCLs have been employed in orchid biotechnology and provides in-depth protocols that will allow for the generation of PLBs using TCLs. As PLBs in orchids are deemed somatic embryos, these will be useful for large-scale mass propagation in bioreactors or for long-term storage as synthetic seeds.     

Rapid micropropagation of monopodial orchid hybrid (Aranda Wan Chark Kuan ‘Blue’?×?Vanda coerulea Grifft. ex. Lindl.) through direct induction of protocorm-like bodies from leaf segments

A competent protocol for accelerated plant regeneration system via direct induction of protocorm-like bodies (PLBs) from leaf of orchid hybrid Aranda Wan Chark Kuan ??Blue???×?Vanda coerulea Grifft. ex. Lindl. was developed for the first time to establish a basis for mass production. Using tissue culture technique, the conditions for PLB induction from leaf explants and conversion of PLBs into plantlets were investigated. Leaves were transferred to MS medium containing different types and concentrations of cytokinins (namely, N⁶-benzyladenine, 6-furfurylaminopurine, N-phenyl-N ^??-(1,2,3-thidiazol-5-yl)urea/TDZ and zeatin) for PLB induction. By means of exploring the effects of cytokinins, it was determined that the optimum PLB induction occurred on MS media supplemented with 1.5?mg?l^?1 TDZ; whereby accordingly, PLB induction (with a frequency of 94.8?%) was observed as early as 8?days of culture and an average of 25 PLBs was obtained from 1?cm² leaf segment after 40?days of culture. Variable pressure scanning electron microscopy indicated the different developmental stages of PLBs in detail. Light/stereo microscopic observation showed the maturation of PLBs and gradual formation of shoot and leaf primordia. More than 96?% conversion (with well-developed shoots and roots) was achieved within the next 30?days of culture, when well developed PLBs were transferred in MS medium supplemented with 1?mg?l^?1 BA, 0.5?mg?l^?1 IBA plus 60?mg?l^?1 adenine sulphate. After 60?days of transfer in plastic pots filled with sand and perlite (2:1; v/v) and with charcoal and coconut fibre (1:1; v/v), subsequently, 90?% well-acclimatized plantlets were recovered.     

文心兰原球茎形态发生与可溶性物质及抗氧化酶的关系

以文心兰切花品种'南茜'无菌苗为材料,取其茎尖通过组织培养诱导形成原球茎和幼苗,观察并分析了原球茎各形态发生阶段的特征及其可溶性糖和蛋白质含量、抗氧化酶(POD、CAT和SOD)活性以及相关同功酶(POD、EST和SOD)的变化.结果显示:(1)文心兰原球茎形态发生可分为外植体期、外植体膨大期、愈伤组织期、原球茎形成期、原球茎成熟期、叶鞘伸展期、顶端腋芽发育期及幼苗期8个阶段.(2)可溶性糖和蛋白质含量均在叶鞘伸展期出现最大峰值;POD活性在外植体膨大期、CAT和SOD活性在愈伤组织期分别出现最大峰值.SOD同工酶的2条酶带在愈伤组织期到幼苗期交替出现;EST同工酶在原球茎形成期有2条特异酶带.研究表明,可溶性糖和蛋白质的含量以及POD、CAT、SOD活性的特异变化与文心兰茎尖脱分化及原球茎再分化的实现密切相关,不同类型的同工酶在原球茎同一发生阶段表现出较大差异,EST同工酶的2条特异酶带可作为原球茎形成的标志.     

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.

     

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.     

Alginate-encapsulation, short-term storage and plant regeneration from protocorm-like bodies of Aranda Wan Chark Kuan ‘Blue’?×?Vanda coerulea Grifft. ex. Lindl. (Orchidaceae)

This article demonstrates the single bead alginate-encapsulation and conversion (complete plantlet regeneration) from protocorm-like bodies (PLBs) of Aranda Wan Chark Kuan ??Blue???×?Vanda coerulea Grifft. ex. Lindl. (AV) (a monopodial orchid hybrid) for the first time. PLBs, induced from leaf segments of AV were isolated from in vitro proliferating PLB clusters. Individual PLBs (4?±?1?mm diameter) were encapsulated in calcium alginate beads to manage mass propagation, short-term storage and germplasm sharing. The superior gel matrix for encapsulation was obtained using 3?% sodium alginate and 75?mM calcium chloride (CaCl₂·2H₂O). Highest percentage of germination (98.1?%) and conversion (96.2?%) of encapsulated PLBs (capsules) was obtained on plant growth regulator-free half-strength MS (Murashige and Skoog, Physiol Plant 15:473?C497, 1962) medium. Successful storage of capsules, until 180?days, was achieved at 25?°C under zero-irradiance with germination and conversion frequency of 76.9 and 70.2?%, respectively. Plantlets regenerated from capsules were acclimatized successfully with 92?% survival rate.     

Formation of Protocorm-like Bodies from Root Apices of Catasetum pileatum (Orchidaceae) Cultivated in Vitro. I. Morphological Aspects

Structural changes in root apices of Catasetum pileatum Reichb.f. cultured in vitro, result in the formation of protocorm-likebodies (PLBs). These PLBs, in turn, give rise to seedlings.After 30 d incubation, the positions occupied by xylem and phloemin the vascular tissue, in transverse sections of the upperportions of PLBs, were typical of stem tissue. The vasculatureof the central part of the PLBs resembled the transition regionbetween shoot and root of whole plants, while in the remainingpart of the PLBs, the vascular arrangement was similar to thatfound in roots. Catasetum pileatum, orchid, root apex, protocorm-like body     

Developmental phases and STM expression during Arabidopsis shoot organogenesis

Shoot organogenesis in Arabidopsis thaliana wasstudied with regard to the timing of key developmental phases and expression ofthe SHOOTMERISTEMLESS (STM) gene.Shoot regeneration in the highly organogenic ecotype C24 was affected byexplanttype and age. The percentage of C24 cotyledon explants producing shootsdecreased from 90% to 26% when donor seedlings were more than 6 dold, but 96% of root explants produced shoots regardless of the age of thedonorplant. Using explant transfer experiments, it was shown that C24 cotyledonexplants required about 2 days to become competent and another 8-10 days tobecome determined for shoot organogenesis. A C24 line containing the promoterofthe SHOOTMERISTEMLESS (STM) genelinked to the -glucuronidase(GUS) gene was used as a tool for determining the timingofde novo shoot apical meristem (SAM) development incotyledon and root explants. Cotyledon and root explants from anSTM:GUS transgenic C24 line were placed on shoot inductionmedium and GUS expression was examined after 6-16 days ofculture. GUS expression could be found in localizedregionsof callus cells on root and cotyledon explants after 12 days indicating thatthese groups of cells were expressing the STM gene, hadreached the key time point of determination, and were producing an organizedSAM. This was consistent with the timing of determination as indicated byexplant transfer experiments. Root explants from anSTM:GUStransgenic Landsberg erecta line and a two-step tissue culture method revealedasimilar pattern of localized GUS expression duringde novo shoot organogenesis. This is the first studydocumenting the timing and pattern of expression of theSTMgene during de novo shoot organogenesis.     

In vitro propagation of <Emphasis Type="Italic">Paphiopedilum</Emphasis> orchid through formation of protocorm-like bodies

Paphiopedilum orchids are among the world’s most popular orchid due to their impressively beautiful flowers. Propagation of these orchid genera has been hampered by the naturally slow growth rate of the plant, which renders it very difficult to be propagated through conventional methods. In vitro culture techniques have provided a useful alternative technology for propagating this recalcitrant species. In this study, the propagation of P. rothschildianum was achieved through the in vitro formation of secondary protocorm-like bodies (PLBs) from the primary PLB that developed from stem-derived callus. The PLBs were cultured on half-strength MS medium supplemented with different concentrations (1.0, 2.0, 3.0, and 4.0 μM) of 6-benzyladenine (BA) and kinetin for the induction of secondary PLBs. The highest number of secondary PLBs formed was obtained on half-strength MS medium supplemented with 4.0 μM kinetin, with an average of 4.1 PLBs per explant after 8 weeks of culture. The secondary PLBs continued to proliferate further and formed 9.5–12.1 new PLBs per secondary PLB after being subcultured onto half-strength plant growth regulator-free MS medium supplemented with 60 g/L banana homogenate (BH). These tertiary PLBs were subcultured onto media containing different organic additives, such as BH, coconut water, potato homogenate, and tomato homogenate, for plantlet regeneration. Among the organic additives tested, the addition of 20% CW to half-strength MS medium resulted in the best average plantlet regeneration percentage from the PLBs, 67.9%, after 8 weeks of culture.     

Direct induction of protocorm-like bodies from shoot tips, plantlet formation, and clonal fidelity analysis in Anthurium andreanum cv. CanCan

Protocorm-like bodies (PLBs) were induced directly at high frequency from wounded surface of Anthurium andreanum cv. CanCan shoot tip-ends, used as explants. In order to obtain PLB directly, the influence of different types and concentrations of cytokinins were evaluated. Amid the cytokinins, N⁶-(?²-isopentenyl)-adenine (2-iP) at a concentration of 15?μM was most effective in inducing PLB whereby ~98 (97.8)?% of explants induced PLB with an average of 120 PLBs per shoot tip within 50?days of culture. Stereomicroscopic observation meticulously revealed the sequential changes from initiation to maturation of PLB gradually forming shoot apical meristem, shoot primordia and leaf primordia. Mature PLBs showed significant shoot proliferation (98.4?%) in media containing 10?μM 6-furfurylaminopurine forming 17 shoots per PLB within 30?days. The inclusion of activated charcoal (AC) in media containing auxin had promotive effect on rooting whereby 5?μM indole-3-butyric acid plus 500?μM AC resulted in highest number and length of roots. Successfully acclimatized plants, subjected to random amplified polymorphic DNA assessment for genetic fidelity, did not show any variation. Thus, this complete study has successfully outlined a rapid, high frequency direct induction of PLB of Anthurium from shoot tips inclusive of shoot proliferation, rooting and acclimatization.     

Multiple Regeneration Pathways via Thin Cell Layers in Hybrid Cymbidium (Orchidaceae)

Protocorm-like body (PLB) and subsequent shoot development in hybrid Cymbidium Twilight Moon ‘Day Light’ can be established in vitro via 3 pathways: PLBs, PLB thin cell layers (TCLs), or embryogenic callus (EC). Traditionally Cymbidium hybrids are mass-produced commercially through the neo-formation of secondary PLBs (2° PLB) from initial or primary PLBs (1° PLB) or PLB segments, or from PLB TCLs, resulting in a moderate number of 2° PLBs (average 4.46 2° PLBs/1° bisected PLB, or 1.12 2° PLBs/ PLB TCL). This study shows that EC can be induced from 1° PLBs or PLB TCLs. Thereafter, resulting 2° PLBs (average 22.1 2° PLBs/EC cluster derived from 1° PLB) form directly from the EC on the same medium or following the transfer of EC onto PGR-free medium. By flow cytometry and PCR-RAPD analysis, the cytogenetic stability of 1° PLBs, of resulting 2° PLBs and EC, and plants derived therefrom was demonstrated.     

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.     

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     

Rapid in vitro propagation of the threatened endemic orchid, Ipsea malabarica (Reichb.f.) J D Hook through protocorm-like bodies

Rapid propagation of I. malabarica (Reichb. f.) J D Hook, an endemic and endangered orchid of the Western Ghats of Kerala, India through conversion of axillary buds to protocorm-like bodies (PLBs), and subsequent plant regeneration was achieved. Growth regulators and sugar displayed significant influence in the induction of PLBs. In vitro derived shoots from field grown rhizomes of Ipsea cultured on Murashige and Skoog (MS) medium with 13.3 microM N6-benzyladenine (BA) containing 2% commercial grade sugar turned the axillary buds to PLBs within 25 days, and developed a mean of 33.1 PLBs within 50 days. Kinetin (KIN) did not induce PLBs, but facilitated axillary bud proliferation. Transfer of PLBs on medium having same concentration of BA and sugar facilitated rapid multiplication, and developed a mean of 47.5 PLBs. No decline of PLB proliferation was observed up to 10th subculture. Half strength MS medium with 6.97 microM KIN facilitated conversion of 98% PLBs to plantlets. On this media, a mean of 5.8 roots were also developed per shoot. Shoots developed bulbs during culture were grown to rhizomes. Increase of sugar to 6 or 8% hastened the development of bulbs/rhizomes. Reintroduction of PLB-derived plantlets in the natural habitat i.e. at Vellarimala (at 1300 m height) of the Western Ghats of Kerala was attempted as a means to assist in situ conservation. This is the first report of conversion of axillary buds to PLBs. The protocol enables to surmount the threat of extinction of this endemic and endangered orchid.     

Expression of SHOOT MERISTEMLESS, WUSCHEL, and ASYMMETRIC LEAVES1 Homologs in the Shoots of Podostemaceae: Implications for the Evolution of Novel Shoot Organogenesis

Podostemaceae (the river weeds) are ecologically and morphologically unusual angiosperms. The subfamily Tristichoideae has typical shoot apical meristems (SAMs) that produce leaves, but Podostemoideae is devoid of SAMs and new leaves arise below the base of older leaves. To reveal the genetic basis for the evolution of novel shoot organogenesis in Podostemaceae, we examined the expression patterns of key regulatory genes for shoot development (i.e., SHOOT MERISTEMLESS (STM), WUSCHEL (WUS), and ASYMMETRIC LEAVES1/ROUGH SHEATH2/PHANTASTICA (ARP) orthologs) in Tristichoideae and Podostemoideae. In the SAM-mediated shoots of Tristichoideae, like in model plants, STM and WUS orthologs were expressed in the SAM. In the SAM-less shoots of Podostemoideae, STM and WUS orthologs were expressed in the initiating leaf/bract primordium. In older leaf/bract primordia, WUS expression disappeared and STM expression became restricted to the basal part, whereas ARP was expressed in the distal part in a complementary pattern to STM expression. In the reproductive shoots of Podostemoideae with a normal mode of flower development, STM and WUS were expressed in the floral meristem, but not in the floral organs, similar to the pattern in model plants. These results suggest that the leaf/bract of Podostemoideae is initiated as a SAM and differentiates into a single apical leaf/bract, resulting in the evolution of novel shoot-leaf mixed organs in Podostemaceae.     

An efficient direct induction of protocorm-like bodies from leaf subepidermal cells of Doritaenopsis hybrid using thin-section culture

High-frequency protocorm-like body (PLB) formation directly from thin leaf sections of Doritaenopsis hybrid was achieved in order to develop a mass-scale propagation system. Concentrated efforts were made to study the effects of different cytokinins on in vitro PLB induction from thin leaf sections. Among the cytokinins tested, thidiazuron (TDZ) was found to be a more effective inducer of PLBs than benzyladenine and zeatin. A modified Murashige and Skoog medium supplemented with 9.0 µM TDZ was found to be the optimum concentration for PLB development from thin leaf sections of Doritaenopsis hybrid. Of the two different explant types used in the present experiment, the highest percentage of PLB formation (72.3%) and highest number of PLBs (18) per explant were observed on thin leaf sections (1 mm thick), while only 20% (4.3 per explant) of comparatively large leaf segments (5 mm thick) were able to produce PLBs under the same culture conditions. Light microscopy observations indicated that the initial cell divisions for PLB formation occurred on the region near the cut surface and that an intact epidermal layer appeared to play an important role in PLB formation. Proembryo initiation occurred from several cells just beneath the intact epidermal cell, and globular PLBs were clearly visible after 3 weeks of culture and subsequently developed into mature PLBs.     

An in vitro method for rapid regeneration of a monopodial orchid hybrid Aranda Deborah using thin section culture

A thin section culture system for rapid regeneration of the monopodial orchid hybrid Aranda Deborah has been developed. Thin sections (0.6–0.7mm thick) obtained by transverse sectioning of a single shoot tip (6–7mm), when cultured in Vacin and Went medium enriched with coconut water (20% v/v), produced an average 13.6 protocorm-like bodies (PLB) after 45 days, compared to 2.7 PLB formed by a single 6–7 mm long shoot tip under same culture condition. Addition of -naphthaleneacetic acid to Vacin and Went medium enriched with coconut water further increased PLB production by thin sections. PLB developed into plantlets on solid Vacin and Went medium containing 10% (v/v) coconut water and 0.5 g l^–1 activated charcoal. With this procedure, more than 80,000 plantlets could be produced from thin sections obtained from a single shoot tip in a year as compared to nearly 11,000 plantlets produced by the conventional shoot tip method.Abbreviations BA 6-benzyladenine - CD callus development - CW coconut water - KC Knudson C medium - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PLB protocorm-like body - TS thin section - VW Vacin and Went medium