In vitro induction and proliferation of protocorm-like bodies (PLBs) from leaf segments of <Emphasis Type="Italic">Phalaenopsis bellina</Emphasis> (Rchb.f.) Christenson

An in vitro culture procedure was established to induce protocorm-like bodies (PLBs) from leaf segments of the Phalaenopsis bellina (Rchb.f.) Christenson directly from epidermal cells without intervening callus on ½ strength modified Murashige and Skoog (MS) (in Physiol Plant 15:473–497, 1962) medium supplemented with 1-Naphthaleneacetic acid (NAA; 0, 0.1, 1 mg/l) and Thidiazuron (TDZ; 0, 0.1, 1, 3 mg/l). The best response was established at 3 mg/l TDZ which induced 78% of leaf segments to form a mean number of 14 PLBs per explant after 16 weeks of culture. No PLBs were found when leaf segments were cultured on ½ strength modified MS media supplemented with 0.1 and 1 mg/l NAA. The best induction percentage for auxin: cytokinin combination was at the combination of NAA and TDZ at 1.0 and 3.0 mg/l which gave 72% induction with 9 PLBs per explant. Semi-solid ½ strength MS and liquid Vacin and Went (VW) (in Bot Gaz 110:605–613, 1949) medium were used in order to find the highest survival and number of PLBs proliferation after 3 months in culture. Half strength MS showed an average of 9 PLBs in comparison with VW with an average of 5.3 PLBs per explants. Histological observations revealed that the regenerated PLBs were generally formed from the epidermal layers of the posterior regions of the leaf segments. Scanning electron micrograph of PLBs showed the origin of newly formed PLB from the peripheral region of leaf segments.     

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

文心兰原球茎液体增殖培养研究

以茎尖诱导形成的原球茎(protocorm-like bodies,PLBs)为外殖体,采用液体培养方式比较了不同浓度的激素配比、蔗糖浓度和添加不同量的新鲜椰汁对文心兰PLBs增殖的影响,并比较了相同培养基成分时液体培养PLBs增殖、分化成苗和固体培养PLBs增殖和分化成苗的差异。试验结果表明:不同浓度的外源激素及其配比对文心兰PLBs增殖生长影响较大,6-BA0.5 mg/L Ad 0.05 mg/L NAA0.05 mg/L的激素组合比较适合文心兰PLBs增殖;蔗糖浓度对文心兰PLBs增殖的影响也较大,适合文心兰PLB在液体培养条件下增殖的蔗糖浓度为7.5 g/L;添加5%新鲜椰汁不仅对文心兰PLBs增殖有促进作用,而且能改善PLBs的质量;适合文心兰PLBs增殖的培养基为MS 6-BA0.5 mg/L Ad 0.05 mg/L NAA0.05 mg/L 5%椰汁 蔗糖7.5 g/L。文心兰PLBs在5周内的增殖生长曲线呈倒"V"字形,第3周的增殖速度达最高峰,而固体培养基PLBs增殖速度较慢,生长曲线几乎成直线。液体增殖的PLBs分化成苗较固体培养增殖的PLBs差。     

Micropropagation of the Thai orchid <Emphasis Type="Italic">Grammatophyllum speciosum</Emphasis> blume

Protocorm-like bodies (PLBs) were induced from shoot tips of Grammatophyllum speciosum, a Thai orchid. The highest frequency of PLBs (93%) were observed on explants incubated on 1/2-Murashige and Skoog (MS) liquid medium containing 2% (w/v) sucrose without any plant growth regulators (PGRs). Tests with different carbon sources compared to sucrose revealed that maltose promoted the highest relative growth of G. speciosum PLBs (7-fold increase), while trehalose and sucrose yielded 5-fold and 4-fold increases, respectively. In 1/2 MS liquid medium, addition of 15 mg/l of chitosan promoted a 7-fold increase in PLB growth while 25 mg/l promoted a 4-fold increase. However, the relative growth rate in solid culture was significantly lower than that in liquid culture. In addition, chitosan supplementation in solid medium promoted shoot formation but not rooting. Plantlet regeneration was induced using a combination of NAA and BA supplementation in 1/2 MS solid medium with optimum induction shoot and root formation at 2.0 mg/l NAA and 1.0 mg/l BA. Using this protocol, approximately 8 months was required to obtain a hundred plantlets from one shoot tip. The plantlets showed no changes in ploidy when tested by flow cytometry.     

Use of protocorm-like bodies for studying alkaloid metabolism in <Emphasis Type="Italic">Pinellia ternata</Emphasis>

Highly differentiated tissue masses known as protocorm-like bodies (PLBs) have been commonly used for plant regeneration. In this study the potential use of PLBs for studying alkaloid metabolism in the Chinese medicinal herb Pinellia ternata (Thunb.) Breit. was investigated. Tuber, leaf, and petiole explants of P. ternata were incubated on Murashige and Skoog (MS) (1962) basal medium containing different combinations of α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA). It was observed that 0.5 mg/L NAA and 1.0 mg/L BA induced the highest frequency of undifferentiated PLBs from tuber explants; whereas, a combination of 0.2 mg/L NAA and 1.0 mg/L BA was best suited for inducing undifferentiated PLBs from leaf and petiole explants. When these PLBs were subcultured on solid MS medium containing 0.6 or 1.2 mg/L abscisic acid (ABA), ABA promoted proliferation of PLBs, but inhibited their germination. To elicit alkaloid biosynthesis, suspension cultures of PLBs were established in half-strength MS (1/2 MS) liquid medium supplemented with 0.6 mg/L ABA. Water extracts of PLBs collected from suspension cultures contained guanosine and inosine, two important alkaloids of P. ternata. Levels of guanosine concentrations were tenfold higher in tuber-derived PLBs compared to those in field-grown tubers; whereas, those of inosine were slightly lower in PLBs compared to those from field-grown tubers.     

Efficient plant regeneration from cotyledon explants of bottle gourd (<Emphasis Type="Italic">Lagenaria siceraria</Emphasis> Standl.)

Using cotyledon explants excised from seedlings germinated in vitro, an efficient plant regeneration system via organogenesis was established for bottle gourd (Lagenaria siceraria Standl.). Maximum shoot regeneration was obtained when the proximal parts of cotyledons from 4-day-old seedlings were cultured on MS medium with 3 mg/l BA and 0.5 mg/l AgNO₃ under a 16-h photoperiod. After 3–4 weeks of culture, 21.9–80.7% of explants from the five cultivars regenerated shoots. Adventitious shoots were successfully rooted on a half-strength MS medium with 0.1 mg/l IAA for 2–3 weeks. Flow cytometric analysis revealed that most of the regenerated plants derived from culture on medium with AgNO₃ were diploid.     

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.     

大薯类原球茎的离体诱导及再生体系的建立

采用正交试验设计方法,以大薯带节茎段为外植体,离体诱导类原球茎并建立大薯类原球茎的再生体系,以解决愈伤组织分化成苗和试管苗移栽成活率低的难题。结果表明：以带节茎段为外植体诱导类原球茎的最适培养基为MS（含3×Ca2＋）＋1.0 mg·L-1 6-BA＋0.2 mg·L-1 NAA＋0.1%PVP＋3%蔗糖,诱导率高达93.33%;类原球茎增殖的最适培养基为MS＋4mg·L-1 6-BA＋80 mg·L-1 Ad＋0.1%PVP＋3%蔗糖;类原球茎生根的最适培养基：1/2MS＋0.10 mg·L-1 NAA＋0.1%PVP＋3%蔗糖。将诱导得到的生根类原球茎植株进行炼苗,移栽基质珍珠岩：蛭石=2：1,移栽成活率可达到95%。     

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.     

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     

The Promntive Effects of Ginseng on Rapid C1onal In Vitro Propagation of African Violet (Saintapulia tonantha Wendl.)

1. Leaf and petiole explants of 31 varielies of African Violet can be induced to regenerate plantlet on MS+0.1 mg/l BA+0.1 mg/l NAA agar medium within 4–6 weeks. 2. Instead of using MS hormone free agar medium, further culturing the explants in MS+ginseng root powder 250 mg/l liquid medium for another 4 weeks has improved the quantity and quality of the regenerated phmtlet. 3. A rapid elonal in vitro propagation protocol was described. The number of transplantable plantlet obtainedby this method was 100–200% more than that obtained by ordinary method. 4. The organogenesis of leaf explants of African Violet was examined by using scanning electron microscopy. The result was described and discussed.     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

Direct shoot organogenesis and assessment of genetic stability in regenerants of <Emphasis Type="Italic">Solanum aculeatissimum</Emphasis> Jacq.

A simple and efficient procedure was developed for in vitro propagation of Solanum aculeatissimum Jacq. using leaf and petiole explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). Effects of various plant growth regulators, explant types, carbohydrates, and basal salts on induction of adventitious shoots were also studied. Leaf explants appeared to have better regeneration capacity than petiole explants in the tested media. The highest regeneration frequency (79.33 ± 3.60%) and shoot number (11.33 ± 2.21 shoots per explant) were obtained in leaf explants in MS medium containing 3% sucrose and 0.8% agar, supplemented with 0.1 mg/l NAA and 2.0 mg/l BA, whereas petiole explants were more responsive to 0.1 mg/l NAA and 1.0 mg/l thiadiazuron. Developed shoots rooted best on MS medium with 1.0 mg/l indole acetic acid (IAA), producing 18.33 ± 2.51 roots per shoot. Histological investigation showed that the shoot buds originated mainly from epidermal cells of wounded tissues, without callus formation. The regenerated plantlets were successfully acclimatized in a greenhouse, where over 90% developed into morphologically normal and fertile plants. Results of flow cytometry analysis on S. aculeatissimum indicated no variation in the ploidy levels of plants regenerated via direct shoot formation and showed almost the same phenotype as that of mother plants. This adventitious shoot regeneration method may be used for large-scale shoot propagation and genetic engineering studies of S. aculeatissimum.     

Shoot regeneration and somatic embryogenesis from different explants of Brahmi [Bacopa monniera (L.) Wettst.]

The morphogenetic potential of node, internode and leaf explants of Brahmi [Bacopa monniera (L.) Wettst.] was investigated to develop reliable protocols for shoot regeneration and somatic embryogenesis. The explants were excised from shoots raised from axillary buds of nodal explants cultured on Murashige and Skoog (MS) basal medium. Presence of 6-benzylaminopurine (BA) or kinetin influenced the degree of callus formation, from which a large number of shoot buds regenerated. Leaf explants gave the largest number of shoot buds followed by node and internode explants. BA was superior to kinetin; BA at 1.5 – 2.0 mg/l appeared to be optimum for inducing the maximum number of shoot buds. MS + 0.1 mg/l BA + 0.2 mg/l indole-3-acetic acid was the most suitable for shoot elongation. Elongated shoots were rooted on full- or half-strength MS medium with or without 0.5 – 1.0 mg/l indole-3-butyric acid or 0.5 – 1.0 mg/l α-naphthaleneacetic acid. The rooted plants were successfully established in soil. Calli derived from nodal explants cultured on MS medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), when subcultured on MS medium containing 0.1 or 0.5 mg/l BA or 0.2 mg/l 2,4-D + 0.1 or 0.5 mg/l kinetin, developed somatic embryos. The somatic embryos germinated either on the same media or on MS basal medium, and the resulting plantlets were successfully transplanted to soil. Received: 25 September 1996 / Revision received: 23 October 1997 / Accepted: 12 November 1997     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

Enhancement of growth and regeneration efficiency from embryogenic callus cultures of Oncidium ‘Gower Ramsey’ by adjusting carbohydrate sources

The embryogenic callus was induced from shoot apex tissues of Oncidium ‘Gower Ramsey’, and the derived callus cultures maintained more than 5 years were viable in growth and exhibited high regeneration capability. Combination levels of exogenous 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ) could stepwise change granular and yellow callus into more friable or compact morphotypes. In the 16-h photoperiod culture, the influences of various carbohydrate sources including sucrose, maltose and trehalose were assessed on formation and development of protocorm-like body (PLB) from the embryogenic callus. Histological observations showed a unicellular origin for these PLBs. The growth of plantlets regenerated on half-strength Murashige and Skoog (MS) medium supplemented with maltose or trehalose was significantly better than those regenerated on sucrose. Approximately, 6000 PLBs could be generated in 2 months from an initial culture of 1 g callus fresh weight, and then more than half of the PLBs developed into plants in 4 months after two subcultures on the medium supplemented with 20 g/l trehalose.     

Artemisinin production by shoot regeneration of Artemisia annua L. using thidiazuron

An efficient in vitro method for multiple shoot bud induction and regeneration has been developed in Artemisia annua L. using leaf and stem explants in various concentrations and combinations of plant growth regulators to evaluate the frequency of regeneration. The sources of explants as well as plant growth regulators in the medium were found to influence the multiple shoot induction. The result shows that the stem segment cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l thidiazuron (TDZ) gave a perfect shoot formation (100%) and good shoot multiplication (57 shoots/explant) after 2 weeks of culture. Healthy regenerated shoots were elongated and rooted in MS medium without hormones. The artemisinin content in plants regenerated from stem explants using 0.1 mg/l TDZ was (3.36 +/- 0.36) microg/mg dry weight and two-fold higher than that of in vitro grown plants of the same age [(1.73 -/+ 0.23) microg/mg DW]. This system exhibited a potential for a rapid propagation of shoots from the stem explant and makes it possible to develop a clonal propagation of A. annua.     

High-frequency direct shoot regeneration from <Emphasis Type="Italic">Drymaria cordata</Emphasis> Willd. leaves

An efficient and reproducible procedure is described for direct shoot regeneration in Drymaria cordata Willd. using leaf explants cultured on Murashige and Skoog (MS) medium supplemented with α-naphthalene acetic acid (NAA) and 6-benzyladenine. The regeneration frequency varied with the plant growth regulator concentrations, orientation of the explants, and the carbon source and basal salts present in the regeneration medium. The highest mean number of shoots per explant (10.65 ± 1.03) was recorded on MS plates containing 3% sucrose and 0.8% agar supplemented with 0.1 mg/l NAA and 1.0 mg/l BAP. Shoot buds were induced in the basal parts of the leaf explants. Concentrations of NAA exceeding 1 mg/l suppressed shoot regeneration. Explants bearing the entire lamina and petiole were much more responsive than those having only the lamina. The plantlets that regenerated from the leaf explants were rooted successively on MS medium alone or in combination with indole butyric acid (IBA). The highest mean number of root organogenesis, with 25.67 ± 3.68 roots per leaf segment, was obtained in the presence of 1 mg/l IBA. Histological investigations of the regenerating shoots showed that the shoot buds had emerged from epidermal cells without callus formation. More than 90% of the in vitro-propagated plants survived when transferred to a greenhouse for acclimatization. Thus, this optimized regeneration system may be used for rapid shoot proliferation and genetic transformation.     

Rapid micropropagation of Geodorum densiflorum (Lam) Schltr. in liquid culture

Technique for rapid mass propagation of Geodorum densiflorum (Lam)Schltr. has been developed by using thin sections of stems of in vitro regenerated plantlets as explant source. Thin sections of stems (1.0-1.5 mm) when cultured in modified liquid and semisolid Knudson C (KnC) medium produced 1.8 and 1.2 protocorm like bodies (PLBs) per explant respectively. Peptone (2 g l-1) was effective in promoting the survival percentage of the explants but had no effect on PLB production. BAP and NAA when used individually enhanced the rate of PLB production. But a significant and manifold increase in PLB production was noted when BAP (3 mg l-1) and NAA (0.5 mg l-1) in combination were added to peptone supplemented liquid and semisolid KnC medium. PLBs thus obtained were subcultured in semisolid KnC medium and obtained well developed plantlets within 10-12 weeks.     

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.