Somatic embryogenesis,organogenesis and plant regeneration in taro (<Emphasis Type="Italic">Colocasia esculenta</Emphasis> var. <Emphasis Type="Italic">esculenta</Emphasis>)

Callus was initiated in three different “esculenta” taro cultivars by culturing corm slices in the dark on half-strength MS medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 20 days followed by subculture of all corm slices to half-strength MS medium containing 1.0 mg/l thidiazuron (TDZ). Depending on the cultivar, 20–30% of corm slices produced compact, yellow, nodular callus on media containing TDZ. Histological studies revealed the presence of typical embryogenic cells which were small, isodiametric with dense cytoplasms. Somatic embryos formed when callus was transferred to hormone-free medium and ~72% of the embryos germinated into plantlets on this medium. Simultaneous formation of roots and shoots during germination, and the presence of shoot and root poles revealed by histology, confirmed that these structures were true somatic embryos. Plants derived from somatic embryos appeared phenotypically normal following 2 months growth in a glasshouse. This method is a significant advance on those previously reported for the esculenta cultivars of taro due to its efficiency and reproducibility.     

Somatic embryogenesis of <Emphasis Type="Italic">Merwilla plumbea</Emphasis> (Lindl.) Speta

A simple and efficient system was developed for rapid somatic embryogenesis from leaf explants of Merwilla plumbea, a traditional but threatened medicinal plant in South Africa. Friable embryogenic callus (FEC) was obtained from leaf explants on embryogenic callus induction medium containing agar-solidified Murashige and Skoog (MS) salts and vitamins, 8.3 μM picloram, 2.3 μM thidiazuron (TDZ) and 20 μM glutamine. FEC was subsequently incubated in embryogenic callus proliferation medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.1 μM picloram for 7 days before it was transferred to liquid somatic embryo medium (SEM_L) containing MS medium supplemented with 0.4 μM picloram and 0.9 μM TDZ. In SEM_L supplemented with 150 mg L⁻¹ haemoglobin, 5.4–35.6 somatic embryos per settled cell volume of 500 mg FEC were obtained. These embryos were at globular to cotyledonary developmental stages. Embryo maturation, germination and plant formation rate was 94.4% following transfer of SEs to half-strength MS medium supplemented with 1.4 μM gibberellic acid. Plantlets transferred into soil acclimatized in the misthouse and established successfully in the greenhouse (100%). This is the first report on induction of Merwilla plumbea somatic embryogenesis. The protocol developed offers controlled vegetative propagation by alleviating extinction threats, ensures germplasm conservation and provides a system for physiological, biochemical, molecular and cellular studies of embryo development.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the rare medicinal plant <Emphasis Type="Italic">Ceropegia candelabrum</Emphasis> L. through somatic embryogenesis

Summary Efficient in vitro propagation of Ceropegia candelabrum L. (Asclepidaceae) through somatic embryogenesis was established. Somatic embryogenesis depended on the type of plant growth regulators in the callus-inducing medium. Friable callus, developed from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 4.52μM2,4-dichlorophenoxyacetic acid (2,4-D), underwent somatic embryogenesis. Compared to solid media, suspension culture was superior and gave rise to a higher number of somatic embryos. Transfer of the friable callus developed on MS medium containing 4.52μM 2,4-D to suspension cultures of half- or quarter-strength MS medium with lower levels of 2,4-D (0.23 or 0.45 μM) induced the highest number of somatic embryos, which developed up to the torpedo stage. Somatic embryogenesis was asynchronous with the dominance of globular embryos. About 100 mg of callus induced more than 500 embryos. Upon transfer to quarter-strength MS agar medium without growth regulators, 50% of the somatic embryos underwent maturation and developed into plantlets. Plantlets acclimatized under field conditions with 90% survival.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration via somatic embryogenesis through cell suspension cultures of horsegram [<Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> (Lam.) verdc.]

Summary In vitro regeneration of plants via somatic embryogenesis through cell suspension culture was achieved in horsegram. Embryogenic calluses were induced on leaf segments on solid Murashige and Skoog (MS) medium with 9.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Differentiation of somatic embryos occurred when the embryogenic calluses were transferred to liquid MS medium containing 2,4-D. Maximum frequency (33.2%) of somatic embryos was observed on MS medium supplemented with 7.9 μM 2,4-D. Cotyledonary-torpedo-shaped embryos were transferred to liquid MS medium without growth regulators for maturation and germination. About 5% of the embryos germinated into plants, which grew further on solid MS medium. The plants were hardened and established in soil. Effects of various auxins, cytokinins, carbohydrates, amino acids, and other additives on induction and germination of somatic embryos were also studied. A medium supplemented with 7.9 μM 2,4-D, 3.0% sucrose, 40 mg l⁻¹ L-glutamine, and 1.0 μM abscisic acid was effective to achieve a high frequency of somatic embryo induction, maturation, and further development.     

Large-scale somatic embryogenesis and regeneration of <Emphasis Type="Italic">Panax notoginseng</Emphasis>

An efficient system for inducing somatic embryogenesis in Panax notoginseng was established using shaker flasks and bioreactor cultures; furthermore, regenerated plantlets were successfully transferred to ex vitro soil conditions. Embryogenic callus was induced from segments of adventitious roots incubated on Murashige and Skoog (MS) medium containing 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) after 5 weeks of culturing. The highest frequency (100%) of somatic embryogenesis, with a mean of 32.7 somatic embryos per callus, was obtained on embryogenic callus incubated on a medium containing 0.5 mg/L 2,4-D. To scale-up somatic embryo formation, 10 g (~1.65 × 10⁴) of early globular-stage somatic embryos were incubated in a 3 L airlift bioreactor containing 1.5 L 1/2 MS medium without plant growth regulators (PGRs) for a period of 4 weeks; these globular-stage somatic embryos then developed into cotyledonary embryos. When maintained on PGR-free medium, the cotyledonary embryos developed roots but did not develop shoots. However, when they were treated with gibberellic acid (GA₃), they continued to germinate and transformed into plantlets after 2 weeks of culture. Plantlets with well-developed shoots and roots were transferred to an autoclaved vermiculite and perlite mixture, acclimatized for a period of 3 months and successfully transferred to forest mountain soil. Following overwintering, these plants produced new growth.     

Somatic embryogenesis and plant regeneration from root sections of <Emphasis Type="Italic">Allium schoenoprasum</Emphasis> L.

A protocol has been developed for somatic embryogenesis and subsequent plant regeneration in Allium schoenoprasum L. Calli were induced from root sections isolated from axenic seedlings and cultivated on media containing either Murashige and Skoog’s (MS) or Dunstan and Short’s mineral solution supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 6-benzylaminopurine (BA), 6-furfurylaminopurine (Kin) or thidiazuron (TDZ) at 1, 5 or 10 μM. The highest frequencies of callus induction were achieved on media with 5 μM 2,4-D in combination with 5 μM TDZ or 10 μM BA (78.9% and 78.4%, respectively). Calli were then transferred to 1 μM 2,4-D, where compact yellow callus turned to segmented yellowish callus with transparent globular somatic embryos at the surface. Calli that were previously grown on media with 5 μM 2,4-D in combination with 10 μM BA or 10 μM TDZ showed the highest frequencies of embryogenic callus formation (45% and 42%) as well as mean number of somatic embryos per regenerating callus. The choice of mineral solution formulation did not significantly affect callus induction or embryogenic callus formation. The embryos could complete development into whole plants on plant growth regulator (PGR)-free medium, but inclusion of Kin (0.5, 2.5 and 5 μM) in this phase improved somatic embryo development and multiplication. Subsequently transferred to 1/2 MS PGR-free medium, all embryos rooted and the survival rate of the plants in a greenhouse was 96%.     

Somatic embryogenesis and plant regeneration in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Embryogenic callus in Catharanthus roseus was initiated from hypocotyl on Murashige and Skoog’s (MS) medium supplemented with 1.0–2.0 mg dm⁻³ of 2,4-dichlorophenoxyacetic acid (2,4-D) or chlorophenoxyacetic acid (CPA). Calli from other sources were non-embryogenic. Numerous somatic embryos were induced from primary callus on MS medium suplemented with naphthalene acetic acid (NAA) within two weeks of culture. Embryo proliferation was much faster on medium supplemented with 6-benzylaminopurine (BAP). After transfer to medium with gibberellic acid (GA₃, 1.0 mg dm^{− 3}) mature green embryos were developed and germinated well into plantlets on MS liquid medium supplemented with 0.5 mg dm⁻³ BAP. Later, embryos with cotyledonary leaves were subjected to different auxins treatments for the development of roots. Before transfer ex vitro, plantlets were cultivated on half strength MS medium containing 3 % sucrose and 0.5 mg dm⁻³ BAP for additional 2 weeks. Additionally, the effect of liquid medium has been evaluated at different morphogenetic stages.     

Effect of plant growth regulators on ovary culture of coconut (<Emphasis Type="Italic">Cocos nucifera</Emphasis> L.)

Coconut is a cross pollinating palm, propagated only by seeds. Tissue culture is the only vegetative propagation method available for coconut. Consistent callogenesis was obtained by culturing unfertilised ovaries at -4 stage in CRI 72 medium containing 100 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1% activated charcoal. Callusing was improved by application of 9 μM thidiazuron (TDZ). Embryogenic calli were subcultured onto somatic embryogenesis induction medium containing 66 μM 2,4-D. Stunted growth was observed in the somatic embryos after subculture onto CRI 72 medium containing abscisic acid (ABA). Maturation of somatic embryos could be achieved in Y₃ medium without growth regulators. Conversion of somatic embryos was induced by adding gibberellic acid (GA₃) to conversion medium containing 5 μM 6-benzyladenine (BA) while 2-isopentyl adenine (2iP) increased the frequency of plant regeneration. A total of 83 plantlets was produced from 32 cultured ovaries.     

Embryogenic responses of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. callus induced from transgenic hairy roots

Agrobacterium rhizogenes A4M70GUS-mediated transformation of two local breeding lines of sugar beet was obtained using 4-week-old seedlings. Root formation efficiency was 61.54% for SBa genotype and 36.36% for SBb genotype. Five highly proliferated hairy root lines have been established in liquid hormone-free MS medium. Transgenic nature of the hairy root clones was evaluated by GUS assay, PCR and RT-PCR analyses. Hairy root-derived calli were induced using different plant growth regulators (PGRs): auxin, auxin/cytokinin and cytokinin. The best callus induction response was achieved on MS medium containing both 1 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg/l thidiazuron (TDZ). Globular embryo-like structures were observed in friable callus after its prolonged cultivation on MS medium supplemented with TDZ and giberellic acid (GA₃) at 1 mg/l each, followed by growth on MS medium containing 1% glucose and 0.5 mg/l 2,3,5-triiodobenzoic acid (TIBA). Histological analysis revealed somatic embryos at different stages of development in hairy root-derived callus of sugar beet.     

Regeneration of <Emphasis Type="Italic">Aeschynanthus radicans</Emphasis> via direct somatic embryogenesis and analysis of regenerants with flow cytometry

Plant regeneration through direct somatic embryogenesis in Aeschynanthus radicans ‘Mona Lisa’ was achieved in this study. Globular somatic embryos were formed directly from cut edges of leaf explants and cut ends or on the surface of stem explants 4 wk after culture on Murashige and Skoog (MS) medium supplemented with N-phenyl-N′-1, 2, 3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA), TDZ with 2,4-dichlorophenoxyacetic acid (2,4-D), or 6-benzylaminopurine (BA) or kintin (KN) with 2,4-D. MS medium containing 9.08 μM TDZ and 2.68 μM 2,4-D resulted in 71% of stem explants producing somatic embryos. In contrast, 40% of leaf explants produced somatic embryos when induced in medium containing 6.81 μM TDZ and 2.68 μM 2,4-D. Somatic embryos matured, and some germinated into small plants on the initial induction medium. Up to 64% of stem explants cultured on medium supplemented with 9.08 μM TDZ + 2.68 μM 2,4-D, 36% of leaf explants cultured on medium containing 6.81 μM TDZ and 2.68 μM 2,4-D had somatic embryo germination before or after transferring onto MS medium containing 8.88 μM BA and 1.07 μM NAA. Shoots elongated better and roots developed well on MS medium without growth regulators. Approximately 30–50 plantlets were regenerated from each stem or leaf explant. The regenerated plants grew vigorously after transplanting to a soil-less substrate in a shaded greenhouse with more than a 98% survival rate. Three months after their establishment in the shaded greenhouse, 500 plants regenerated from stem explants were morphologically evaluated, from which five types of variants that had large, orbicular, elliptic, small, and lanceolate leaves were identified. Flow cytometry analysis of the variants along with the parent showed that they all had one identical peak, indicating that the variant lines, like the parent, were diploid. The mean nuclear DNA contents of the variant lines and their parent ranged from 4.90 to 4.99 pg 2C⁻¹, which were not significantly different statistically. The results suggest that the regenerated plants have a stable ploidy level, and the regeneration method established in this study can be used for rapid propagation of ploidy-stable Aeschynanthus radicans.     

Somatic embryogenesis and shoot organogenesis from leaf explants of <Emphasis Type="Italic">Primulina tabacum</Emphasis>

Primulina tabacum is a rare and endangered species that is endemic to China. Establishing an efficient regeneration system is necessary for its conservation and reintroduction. In this study, when leaf explants collected from plants grown in four ecotypes in China are incubated on Murashige and Skoog (MS) medium containing 5.0 μM thidiazuron (TDZ) for 30 days, then transferred to medium containing 5.0 μM 6-benzyladenine (BA), adventitious shoots are then observed. Conversely, when leaf explants are incubated on medium containing 5.0 μM BA for 30 days, then transferred to medium containing 5.0 μM TDZ, somatic embryogenesis is induced. This indicates that somatic embryogenesis and shoot organogenesis could be switched simply by changing the order of two cytokinins supplemented in the culture medium. Histological investigation has revealed that embryogenic cells are induced within 30 days following incubation of explants in medium containing TDZ. Only if embryogenic cells were induced, TDZ could enhance somatic embryogenesis and BA could stimulate shoot organogenesis. When comparing explants from different ecotypes, leaf explants from Zixiadong in Hunan Province could induce low numbers (1–2) of either somatic embryos or adventitious shoots on medium containing either 5.0 μM TDZ or 5.0 μM BA, respectively. Whereas, leaf explants from plants collected from the other three ecological habitats could induce 50–70 somatic embryos/adventitious shoots per explant. Moreover, somatic embryos could induce secondary somatic embryogenesis and adventitious shoots on different media. All regenerated shoots developed adventitious roots when these are transferred to rooting medium, and over 95% of plantlets have survived following acclimatization and transfer to a potting mixture (1:1, sand:vermiculite).     

Plant regeneration using immature zygotic embryos of <Emphasis Type="Italic">Tribulus terrestris</Emphasis>

The genus Tribulus is the source of a number of steroidal saponins and other bioactive compounds which are of medicinal and pharmaceutical importance and plant regeneration of Tribulus terrestris has been reported. The objective of this study was to evaluate the potential of immature zygotic embryos of Tribulus terrestris as an explant for plant regeneration. Embryos were cultured on MS medium supplemented with 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), alone or in combination and callus and shoot or embryo formation evaluated. With 2.5 mg/l NAA or 2,4-D, callus formation frequency was 100% but 57% with 2.5 mg/l TDZ. The combination of 2.5 mg/l TDZ and NAA or 2,4-D also elicited callus formation frequency of 100%. The callus formation frequency was lower with lower levels of these growth regulators. On a medium with 0.5 mg/l TDZ, 17.4% of the 2,4-D-derived callus (2.5 mg/l), developed embryo-like structures and this increased to 37.3 and 41.4% respectively, when TDZ was combined with 0.5 mg/l indole-3-butyric acid (IBA) or 2,4-D. Both shoot formation and embryo-like structures developed in cultures with 2.5 mg/l TDZ, alone or in combination with 0.5 mg/l IBA or 2,4-D. The optimum sucrose level for morphogenetic response of embryo-derived callus was between 5.0 and 7.5%. Embryo-like structures were also observed when the 2,4-D-derived callus was cultured in a liquid containing benzyladenine (BA) and IBA. Plants were regenerated from both embryo-like structures and shoot buds on solid MS medium containing 0.2 mg/l IBA and rooted plantlets were transferred to soil.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of Persian poppy (<Emphasis Type="Italic">Papaver bracteatum</Emphasis>)

Persian poppy (Papaver bracteatum Lindl.) is an important commercial source of medicinal opiates and related compounds. In this research, calli were induced from seeds, roots, cotyledons and hypocotyls of P. bracteatum at a high efficiency. The optimized callus induction media consisted of the Murashige and Skoog (MS) basic media supplemented with 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L kinetin and 15 mg/L ascorbic acid. The concentrations of 2,4-D and ascorbic acid were found critical to callus induction and proliferation. Subsequent subcultures resulted in excellent callus proliferation. Ascorbic acid at concentration 15 mg/L increased the callus proliferation significantly. Maximum callus growth was achieved when the explants were incubated at 25°C. MS salts at full strength were found inhibitory for callus induction, while ľ MS salts were found to favor callus induction. Shoot regeneration of calli in vitro was achieved on ľ MS medium containing 0.5 mg/L benzylamine purine and 1.0 mg/L naphthalene acetic acid. Analysis of alkaloid extracts from Persian poppy tissues by high-performance liquid chromatography showed that thebaine accumulated in the tissues of plants. The thebaine alkaloid profile of the Persian poppy is a well-defined model to evaluate the potential for metabolic engineering of thebaine production in P. bracteatum.     

Somatic embryogenesis and regeneration of <Emphasis Type="Italic">Vigna radiata</Emphasis>

An efficient regeneration protocol via somatic embryogenesis was optimized for mung bean [Vigna radiata (L.) Wilczek; cv. Vamban 1]. Primary leaf explants were used for embryogenic callus induction in MMS medium (Murashige and Skoog salts with B5 vitamins) containing 2.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D), 150 mg dm⁻³ glutamine and 3 % sucrose. Fast growing, highly embryogenic cell suspensions were established from 21-d-old calli in MMS medium supplemented with 0.5 mg dm⁻³ 2,4-D and 50 mg dm⁻³ proline (Pro), and maximum recovery of globular (39.0 %), heart-shaped (26.3 %) and torpedo-stage (21.0 %) somatic embryos were observed in this medium. Mature cotyledonary-stage somatic embryos were cultured for 5 d in half strength B5 liquid medium containing 0.05 mg dm⁻³ 2,4-D, 20 mg dm⁻³ Pro, 5 μM abscisic acid, 1000 mg dm⁻³ KNO₃, 50 mg dm⁻³ polyethylene glycol (PEG 6000) and 30 g dm⁻³ D-mannitol. Mature somatic embryos were germinated after dessication for 3 d and complete development of plantlets accomplished in MMS medium containing 30 g dm⁻³ maltose, 0.5 mg dm⁻³ benzyladenine and 500 mg dm⁻³ KNO₃. Profuse lateral roots, and regeneration frequency (up to 60 %) were observed in half-strength MMS medium containing 0.5 mg dm⁻³ indolebutyric acid (IBA). The regenerated plants were grown to fruiting and were morphologically normal and fertile.     

Primary and repetitive secondary somatic embryogenesis of <Emphasis Type="Italic">Lepidosperma drummondii</Emphasis> (Cyperaceae) and <Emphasis Type="Italic">Baloskion tetraphyllum</Emphasis> (Restionaceae) for land restoration and horticulture

Somatic embryogenesis was developed as a method of mass propagation for Lepidosperma drummondii (Cyperaceae), a difficult to propagate but important species for post-mining restoration in a region of high plant biodiversity, in the southwest of Western Australia. Cultures were initiated from excised zygotic embryos, shoot cultures to rhizomes. Only zygotic embryos of L. drummondii developed somatic embryos, with half strength Murashige and Skoog basal medium (BM) and 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) being the most effective combination. The first culture cycle yielded a mean of 30 somatic embryos per excised zygotic embryo forming an embryo cluster. After a further 6 wk in culture (on fresh BM with 1 μM 2,4-D), approximately 350 somatic embryos per starting embryo cluster were recorded. Following regular sub-culturing of primary somatic embryo clusters onto fresh media (every 4 wk), more than 74,000 secondary somatic embryos were estimated to have been produced after eight subculture periods. This translates to between 1,000 and 2,000 somatic embryos produced from an estimated 45 mg of starting tissue per culture plate or potentially 22,0000–44,000 somatic embryos per gram of tissue. This is a significant improvement over all previous methods used to propagate L. drummondii, in which typical in vitro shoot multiplication rates are as low as 1.43 per 8 wk. This also compared favourably with published data and concurrent experiments undertaken in this study (as an extra control measure) on somatic embryo production for a related species Baloskion tetraphyllum (using the same BM with 1 μM 2,4-D and coleoptile segments as explants). Various media combinations were investigated for efficacy in converting somatic embryos into plants with best results ranging from 86% to 100% conversion for B. tetraphyllum on BM without plant growth regulators. Development of L. drummondii somatic embryos into plants was not observed on BM without plant growth regulators. However, a best result of 39% conversion to plants was observed on BM with 1 μM thidiazuron. This is the first report of successful development of somatic embryogenesis and conversion of somatic embryos into plants using thidiazuron for the Australian cyperale L. drummondii.     

Somatic embryo productions by liquid shake culture of embryogenic calluses in <Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper

The regeneration of plants via somatic embryogenesis liquid shake culture of embryogenic calluses was achieved in Vigna mungo (L.) Hepper (blackgram). The production of embryogenic callus was induced by seeding primary leaf explants of V. mungo onto Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented (optimally) with 1.5 mg/l 2,4-dichloro-phenoxyacetic acid. The embryogenic callus was then transferred to liquid MS medium supplemented (optimally) with 0.25 mg/l 2,4-dichloro-phenoxyacetic acid. Globular, heart-shaped, and torpedo-shaped embryos developed in liquid culture. The optimal carbohydrate source for production of somatic embryos was 3% sucrose (compared to glucose, fructose, and maltose). l-Glutamine (20 mg/l) stimulated the production of all somatic embryo stages significantly. Torpedo-shaped embryos were transferred to MS (Physiol Plant 15:473–497, 1962) liquid medium containing 0.5 mg/l abscisic acid to induce the maturation of cotyledonary-stage embryos. Cotyledonary-stage embryos were transferred to 1/2-MS semi-solid basal medium for embryo conversion. Approximately 1–1.5% of the embryos developed into plants.     

Somatic embryogenesis and plant regeneration from callus cultures of several species in the genus <Emphasis Type="Italic">Tricyrtis</Emphasis>

Summary The liliaceous perennial plants, Tricyrtis spp., are cultivated as ornamental plants in Japan. Natural populations of several Japanese Tricyrtis spp. are severely threatened by indiscriminate collection and habitat destruction. In this study, a plant regeneration system based on somatic embryogenesis has been developed for efficient clonal propagation of T. hirta, T. hirta var. albescens, T. formosana, T. formosana cv. Fujimusume, T. flava ssp. ohsumiensis, and T. macrantha ssp. macranthopsis. Flower tepal explants of these genotypes were cultured on media containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (picloram, PIC) alone or in combination with N-(1,2,3-thiadiazol-5-yl)-N′-phenylurea (thidiazuron, TDZ). Calluses induced on media containing 2,4-D produced somatic embryos following their transfer to a plant growth regulator-free medium, indicating that these calluses were embryogenic. A combination of 4.5μM2,4-D and 0.45 μM TDZ was most effective for inducing embryogenic calluses from tepal explants. Among various explant sources, filaments were most suitable for inducing embryogenic calluses on a medium containing 4.5μM 2,4-D and 0.45 μM TDZ. Embryogenic calluses were only obtained from filament explants for T. macrantha ssp. macranthopsis. Embryogenic calluses could be maintained by subculturing monthly onto the same medium, and a 1.5–3.5-fold increase in fresh weight was obtained after 1 mo. of subculture. Depending on the plant genotype, 50–500 somatic embryos per 0.5g fresh weight of embryogenic callus was obtained 1 mo. after transfer to a plant growth regulator-free medium. Most of the embryos developed into plantlets, and they were successfully acclimatized to greenhouse conditions. Regenerated plants showed no alteration in the ploidy level as indicated by chromosome observation and flow cytometric analysis.     

A rapid system for micropropagation of <Emphasis Type="Italic">Swertia chirata</Emphasis> Buch-Ham. ex Wall.: an endangered medicinal herb via direct somatic embryogenesis

An improved method of direct somatic embryogenesis (SE) was developed in Swertia chirata for the first time using leaves and roots of in vitro-grown young seedlings. In the present study, 2,4-dichlorophenoxyacetic acid (2,4-D) was assessed individually and in combination with other auxins, as well as with cytokinin for its effectiveness to induce somatic embryos. Leaf explants with abaxial side in the medium produced maximum number of somatic embryos. This system omits the callus stage and thus reduces the process of SE in S. chirata by 35–45 days. Embryos at different stages of development were observed. Maturation of heart stage embryos were observed on Murashige and Skoog (MS) medium containing 1 mg L⁻¹ 2,4-D. Upon transfer to the germination medium, they were converted to cotyledonary stage and then plantlets of 33% and 68% of them were converted to cotyledonary stage and then plantlets on MS medium supplemented with 0.05 and 0.1 mg L^-1 GA₃ respectively. The 2,4-D alone at 1.0 or 1.5 mg L⁻¹ was found to be better for embryogenic tissue initiation than 2,4-D in combination with indole-3-acetic acid or α-naphthalene acetic acid. For further embryo development, 2,4-D was combined with cytokinins such as 6-benzylaminopurine (BAP) and kinetin or plant growth regulator free medium or medium with 50% reduced concentration of the same hormone while subculturing. Mean germination and percentage of survival were maximum in the medium containing 1.0 mg L⁻¹ 2,4-D in combination with 0.1 mg L⁻¹ BAP. Regenerated plantlets were morphologically and genetically identical. This method offers a vast scope for the clonal propagation of endangered plants.     

A reliable protocol for plant regeneration from callus culture of <Emphasis Type="Italic">Phalaenopsis</Emphasis>

Summary Callus of Phalaenopsis Nebula was induced from seed-derived protocorms on 1/2 Murashige and Skoog (MS) basal medium plus 0–1.0 mg l⁻¹ (0–4.52 μM) N-phenyl-N′-1,2,3,-thiadiazol-5-yl urea (TDZ) and/or 0–10 mg l⁻¹ (0–45.24 μ M) 2,4-dichlorophenoxyacetic acid (2,4-D). Protocorms 2 mo. old performed better than 1-mo.-old protocorms for callus induction. More calluses formed on 1/2 MS basal medium supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. These calluses could be maintained by subculturing every month with basal medium supplemented with 0.5 mg l⁻¹ (2.27 μM) TDZ and 0.5 mg l⁻¹ (2.26 μM) 2,4-D. Protocorm-like bodies were formed, and plants regenerated from these calluses on 1/2 MS basal medium alone or supplemented with 0.1–1.0 mg l⁻¹ (0.45–4.52 μM) TDZ. Plantlets were then potted on sphagnum moss in the greenhouse and grew well. No chromosomal abnormalities were found among the root-tip samples of 21 of the regenerated plantlets that were successfully acclimatized.     

Androgenesis in the vine cacti <Emphasis Type="Italic">Selenicereus</Emphasis> and <Emphasis Type="Italic">Hylocereus</Emphasis> (Cactaceae)

Anther culture techniques were applied to develop a methodology for producing haploid vine cactus plants. Anthers with most microspores in the middle uninucleate developmental stage from the tetraploid species Selenicereus megalanthus and the diploid species Hylocereus polyrhizus and H. undatus were cultured on basal MS medium supplemented with picloram and 6-benzyladenine (BA). H. polyrhizus and H. undatus anthers were also cultured on MS medium containing either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid (2,4-D). Pro-embryo development started after three days of culture. A direct androgenic embryo response was achieved in S. megalanthus with and without picloram/BA, while H. polyrhizus exhibited non-regenerative callus formation. Only a single direct androgenic embryo was obtained in H. polyrhizus (with 0.1 mg/l TDZ). H. undatus required a cold pre-treatment of 4°C for 24 h in 0.3 M D-mannitol to produce a response, but only calluses were obtained and they did not regenerate. S. megalanthus and H. polyrhizus embryos converted into plantlets after transfer to MS medium in the light. Rooted plants were acclimatized successfully, and most plants showed normal phenotypes. Flow cytometry and cytological studies revealed monoploid, haploid, dihaploid, and mixoploid plants. This study showed that androgenesis is strongly species- and culture-medium-dependent, thus revealing new perspectives in the genetics and breeding of vine cactus species. To the best of our knowledge, this is the first report on the production of monoploid, haploid and dihaploid plants in Cactaceae.