Ploidy stability of somatic embryogenesis-derived <Emphasis Type="Italic">Passiflora cincinnata</Emphasis> Mast. plants as assessed by flow cytometry

In this study, flow cytometric analysis was used to evaluate the genetic stability of Passiflora cincinnata Mast. plants regenerated via primary and secondary somatic embryogenesis. Embryogenic calli obtained from culturing zygotic embryos on Murashige and Skoog (MS) medium containing 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM benzyladenine (BA) were transferred to differentiation medium. Torpedo and cotyledonary embryos were obtained. These primary embryos were maintained on differentiation medium to generate secondary embryos. Conversion of primary and secondary embryos yielded 305 and 138 normal plants, respectively. Almost 90% of plantlets survived following acclimatization. Flow cytometric analysis revealed that seed-derived plants had on average 3.01 pg nuclear DNA (2C), and all plants, except for a single plant regenerated via primary embryogenesis, maintained their ploidy. This single plant contained more than twice the average DNA content: 6.21 pg (4C). Epidermal stomata of leaves of the tetraploid plant were larger but lower in density than those of diploid plants, indicating that stomatal characteristics are useful in distinguishing between diploid and tetraploid plants of passion fruit. In summary, the procedure we employed to regenerated P. cincinnata plants via somatic embryogenesis generated mostly genetically true-to-type plants.     

A novel regeneration system for a wild passion fruit species (<Emphasis Type="Italic">Passiflora cincinnata</Emphasis> Mast.) based on somatic embryogenesis from mature zygotic embryos

The objective of the present work was to induce somatic embryogenesis from zygotic embryos of Passiflora cincinnata Masters. Zygotic embryos formed calli on media with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.5 μM benzyladenine (BA) after 30 days of in vitro culture. A concentration of 18.1 μM 2,4-D resulted in the largest number of somatic embryos. Embryogenic calli were yellowish and friable, forming whitish proembryogenic masses. Morphologically, embryogenic cells were small and had large nuclei and dense cytoplasm, whereas non-embryogenic cells were elongated, with small nuclei and less dense cytoplasm. Calli cultured under white light on basal Murashige and Skoog’s medium with activated charcoal produced embryos in all developmental stages. There were differences among the treatments, with some leading to the production of calli with embryos and some only to callus formation. Some abnormalities were associated with somatic embryos, including fused axes, fused cotyledons and polycotyledonary embryos. Production of secondary somatic embryos occurred in the first cycle of primary embryo development. Secondary embryos differentiated from the surface of the protodermal layer of primary embryos with intense cell proliferation, successive mitotic divisions in the initial phase of embryoid development, and a vascular system formed with no connection to the parental tissue. This secondary embryogenic system of P. cincinnata is characterized by intense proliferation and maintenance of embryogenic competence after successive subcultures. This reproducible protocol opens new prospects for massive propagation and is an alternative to the current organogenesis-based transformation protocol.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration through somatic embryogenesis and direct shoot organogenesis in <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.

An efficient in vitro plant regeneration protocol through somatic embryogenesis and direct shoot organogenesis has been developed for pearl millet (Pennisetum glaucum). Efficient plant regeneration is a prerequisite for a complete genetic transformation protocol. Shoot tips, immature inflorescences, and seeds of two genotypes (843B and 7042-DMR) of pearl millet formed callus when cultured on Murashige and Skoog (MS) medium supplemented with varying levels of 2,4-dichlorophenoxyacetic acid (2,4-D; 4.5, 9, 13.5, and 18 μM). The level of 2,4-D, the type of explant, and the genotype significantly effected callus induction. Calli from each of the three explant types developed somatic embryos on MS medium containing 2.22 μM 6-benzyladenine (BA) and either 1.13, 2.25, or 4.5 μM of 2,4-D. Somatic embryos developed from all three explants and generated shoots on MS medium containing high levels of BA (4.4, 8.8, or 13.2 μM) combined with 0.56 μM 2,4-D. The calli from the immature inflorescences exhibited the highest percentage of somatic embryogenesis and shoot regeneration. Moreover, these calli yielded the maximum number of differentiated shoots per callus. An efficient and direct shoot organogenesis protocol, without a visible, intervening callus stage, was successfully developed from shoot tip explants of both genotypes of pearl millet. Multiple shoots were induced on MS medium containing either BA or kinetin (4.4, 8.8, 17.6, or 26.4 μM). The number of shoots formed per shoot tip was significantly influenced by the level of cytokinin (BA/kinetin) and genotype. Maximum rooting was induced in 1/2 strength MS with 0.8% activated charcoal. The regenerated plants were transferred to soil in pots, where they exhibited normal growth.     

Production of triploid plants of papaya by endosperm culture

Triploid papaya (Carica papaya) plants were obtained by immature endosperm culture. Visible callusing of the endosperm occurred 21 days after initiation of cultures. A continuously growing callus was observed and a maximum of 68.7% of callus induction frequency was obtained when immature endosperm with embryo was cultured on Murashige and Skoog (MS) medium containing 6.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 2.5 μM α-naphthaleneacetic acid (NAA) and 4.0 μM 6-furfurylamino purine (Kn). Shoot buds were produced when the callus was subcultured on a medium supplemented with 6-benzyladenine (BA) or thidiazuron (TDZ) along with NAA. Shoots were detached from the callus and transferred to the elongation medium supplemented with indole-3-acetic acid (IAA) and BA. The combination of 3.0 μM IAA and 1.5 μM BA was the best in terms of the number of cultures (93.8%) showing axillary shoot proliferation. The addition of 2.0 μM indole-3-butyric acid (IBA) to the 1/2 MS medium was most effective at inducing root formation with 90% of the shoots developing four to five roots. Healthy rooted plantlets were transferred to pots containing sterilized bed soil and perlite (3:1) mixture in the greenhouse and 78% of the micropropagated plants survived transplantation. The leaves from endosperm-derived plants showed larger stomata and more chloroplasts in guard cells than that from the parent plants. Over 75% of the endosperm-derived plants were triploid with chromosome number 2n = 3x = 27.     

Regeneration of <Emphasis Type="Italic">Clivia miniata</Emphasis> and assessment of clonal fidelity of plantlets

An efficient in vitro micropropagation system for Clivia miniata Regel was developed using basal tissues of young petals and young ovaries as explants. For callus induction, explants were incubated on Murashige and Skoog (MS) medium containing either 2.22 μM 6-benzyladenine (BA) and 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 4.44 μM BA, 5.37 μM α-naphthaleneacetic acid (NAA), and 9.05 μM 2,4-D. Moreover, callus was induced from young ovaries when these were incubated on MS medium containing 8.88 μM BA, 10.74 μM NAA, and 9.05 or 18.10 μM 2,4-D. Subsequently, callus was transferred to MS medium supplemented with kinetin (KT) and NAA for shoot organogenesis. Frequency of shoot regeneration from petal-derived callus was highest when callus was transferred to medium containing 2.69 μM NAA with either 9.29 or 13.94 μM KT. Shoot regeneration frequency from ovary-derived callus was highest when this callus was transferred to medium containing 9.29 μM KT and 10.74 μM NAA. Overall, different explant types exhibited different organogenic capacities wherein, young petals had higher shoot regeneration frequencies than young ovaries. The highest rooting frequency (98.25 ± 3.04%) was obtained when shoots were transferred to half-strength MS medium without plant growth regulators. Regenerated plantlets were transplanted to soil mix and acclimatized, yielding a 96.80% survival frequency. Only 0.6% of regenerated plantlets exhibited morphological changes. The diploid status (2n = 22) of regenerated plantlets was determined using chromosome counts of root-tips. Moreover, inter-simple sequence repeats were used to assess the genetic fidelity of regenerated plantlets. Overall, regenerated plants shared 90.5–100.0% genetic similarities with mother plants and 89.0–100.0% similarities with each other.     

Somatic embryogenesis and metabolic differences between embryogenic and non-embryogenic structures in mangosteen

Somatic embryogenesis in mangosteen (Garcinia mangstana L.) was investigated using seed and leaf segments cultured on Murashige and Skoog medium with treatments of 6-benzyladenine (BA) [2.0, 3.0, 4.0 µM] and 2,4-diclorophenoxyacetic acid (2,4-D) [4.5, 9.0, 13.5 µM]. There were four types of structures (globular, nodular compact, friable and spongy) formed. Two treatments resulted in embryogenic characteristics from seed cultures; the highest percentage 46.67?% of globular structure (resembling somatic embryos) grown on 3.0 µM BA and 80?% of nodular compact structures on 4.0 µM BA?+?13.5 µM 2,4-D. For the leaf culture, highest percentage, 93.33?% produced nodular compact structures on 2.0 µM BA?+?4.5 µM 2,4-D. Histological analysis showed that the globular structure has well-defined protoderm and separated from the original explant. Nodular compact structure also showed the presence of densely cytoplasmic meristematic cells with a high nucleoplasmic ratio. These characteristics observed in globular and nodular compact structure indicates somatic embryo formation. The globular structures which were converted into shoots and roots (60.00?%) showed atypical somatic embryogenesis in mangosteen. Metabolite fingerprinting was carried out using gas chromatography–mass spectrometry. Amino acids, carbohydrates, organic acids and fatty acids were found in both the embryogenic structures and non-embryogenic structures tested. Multivariate discriminant analyses of the metabolic data revealed significant metabolites (P?≤?0.05) for both types of structures. Principle component analysis suggested that amino acids and carbohydrates were the major compounds distinguishing embryogenic and non-embryogenic structures. Ornithine and mannose were present at significant level in embryogenic structures as compared to non-embryogenic ones while fructose was significantly higher in non-embryogenic structures.     

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (<Emphasis Type="Italic">Passiflora edulis</Emphasis> Sims) genotypes

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed.     

In vitro propagation of <Emphasis Type="Italic">Cymbidium goeringii</Emphasis> Reichenbach fil. through direct adventitious shoot regeneration

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and thidiazuron (TDZ) on direct rhizome induction and shoot formation from rhizome explants of Cymbidium goeringii was explored. Rhizome segments obtained from in vitro seed cultures of C. goeringii were placed on Murashige and Skoog (MS) medium incorporated with 5, 10, 20, or 40 µM 2,4-D and 1, 2, 4, or 8 µM BA or TDZ alone or in combination with 20 µM 2,4-D. The explants developed only rhizomes on MS medium with or without 2,4-D. The highest percent of rhizome formation (100%) was obtained on MS medium incorporated with 20 μM of 2,4-D. The morphology and number of rhizomes varied with the level of 2,4-D in the medium. Direct adventitious shoot formation was achieved on medium incorporated with BA or TDZ. The adventitious shoots produced per explant significantly increased with the supplementation of 2,4-D to cytokinin-containing medium. The highest mean of 21.8 ± 1.8 shoot buds per rhizome segment was obtained in medium fortified with 20 μM 2,4-D and 2 μM TDZ. The greatest percent of root induction (100%) and the mean of 5.3 ± 1.1 roots per shoot were achieved on ½ MS medium incorporated with 2 μM of α-naphthaleneacetic acid. About 97% of the in vitro-produced plantlets acclimatized in the greenhouse. An efficient in vitro propagation protocol was thus developed for C. goeringii using rhizome explants.     

Somatic embryogenesis and plant regeneration from fragments of immature inflorescences and coleoptiles of durum wheat

Use of Hypericum perforatum L. has increased in the past few years due to the antidepressant and antiviral activities found in extracts of this plant. As a result of its potential as a pharmaceutical, a new system was developed for in vitro culture of this species. Leaf explants were inoculated onto MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D, 0.45 or 4.5 μM) and 6-benzyladenine (BA, 0.44 or 4.4 μM) or kinetin (0.46 or 4.6 μM). Explants were cultivated under dark or light conditions to induce callus formation. Callus initiation was observed in all media evaluated and the highest cell proliferation was obtained from explants cultivated in the presence of 4.4 μM BA and 4.5 μM 2,4-D in the dark. Shoot induction was obtained from callus induced on 4.6 μM kinetin and 0.45 μM 2,4-D 6 weeks after transferring the callus to a MS medium supplemented with 4.4 μM BA. Roots were induced from shoots on full and half-strength MS media with or without indolebutyric acid (IBA, 4.9 μM) and the highest rooting frequencies were obtained on half-strength MS medium, regardless of the presence of IBA. Regenerated plants were easily acclimated in greenhouse conditions. The procedure reported here allows the micropropagation of H. perforatum in five months of culture and the proliferation of cell masses which could be used for studies on organic compounds of pharmaceutical interest. This revised version was published online in June 2006 with corrections to the Cover Date.     

ABA enhances plant regeneration of somatic embryos derived from cell suspension cultures of plantain cv. Spambia (<Emphasis Type="Italic">Musa</Emphasis> sp.)

Creamy friable calli were induced from meristems (scalps) of proliferating shoots of plantain (Musa sp.) cv. Spambia (genome AAB) incubated on a semi-solid modified Murashige and Skoog (MS) medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 μM zeatin. About 25% of shoot-tip explants formed scalps, and about 98% of scalps developed embryogenic calli. Small dense aggregates of cells, were obtained when these calli were transferred to liquid MS medium supplemented with 4.5 μM 2,4-D and 1.0 μM zeatin. Upon transfer to semi-solid MS medium of the same composition as described above, aggregates of cells formed somatic embryos. In the presence of 2.5 μM abscisic acid (ABA), maturation of somatic embryos was 2.6-fold higher than that of control (lacking ABA), and regardless of the type of cytokinin used in the medium. Upon transfer to MS medium supplemented with 1.25 μM 6-benzyladenine (BA), 80% of germinated embryos developed into plantlets.     

Regeneration of zoysia grass (Zoysia matrella L. Merr.) cv. Konhee from young inflorescences and stem nodes

We have optimized conditions for efficient regeneration of the vegetatively propagated zoysia grass (Zoysia matrella L. Merr) cultivar “Konhee”. Two explants, young inflorescences, and stem nodes, were used and they displayed different responses to combinations and concentrations of plant growth regulators in callusing, embryogenic callus formation, and regeneration. The highest callus initiation rate from young inflorescences was obtained on medium supplemented with 4.5 to 9.0 μM 2,4-dicholorophenoxy acetic acid (2,4-D) and 0.44 μM 6-benzyl amino purine (BA). When the BA concentration was lowered to 0.044 μM, the highest percent embryogenic callus induction from young inflorescences was achieved. The highest callus initiation rate from stem nodes was obtained, when young inflorescences were cultured on MS medium supplemented with 4.5 to 9.0 μM 2,4-D, 0.44 μM BA, and 0.037 μM abscisic acid (ABA). But embryogenic callus formation from the stem node was highest in the presence of 4.5 to 9.0 μM 2,4-D, 0.044 μM BA, and 0.037 μM ABA. Addition of ABA significantly increased embryogenic callus formation from stem nodes, but not from young inflorescences. Regeneration percentage was variable in response to BA level, and inclusion of α-naphthalene acetic acid (NAA) and gibberellic acid (GA₃) further increased the regeneration percentage. The highest regeneration percentages obtained from the young inflorescences and stem nodes were 82% and 67%, respectively. This is the first report showing that plants can be regenerated from young inflorescences and stem nodes of vegetatively propagated zoysia grass.     

Plant regeneration through somatic embryogenesis of a medicinal plant, <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr.

Somatic embryogenesis and subsequent plant regeneration were established from hypocotyl and internode explants collected from in vitro-grown seedlings and in vitro-proliferated shoots, respectively. Somatic embryogenesis was significantly influenced by the types of auxin and cytokinin. Friable calluses with somatic embryos developed well in Murashige and Skoog basal (MS) medium supplemented with 0.8–8.8 μM 6-benzylaminopurine (BA) and 2.0–8.0 μM 2,4-dichlorophexoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The maximal frequency of embryogenic callus and somatic embryo formation were obtained when the MS medium was amended with 8.8 μM BA and 4.0 μM 2,4-D. The best embryo germination occurred in a hormone-free 1/2-MS medium. The highest percentage of shoot proliferation was observed in embryogenic calluses in MS medium containing 2.0 μM BA and 1.0 μM NAA. In vitro-grown shoots were rooted in MS medium with 0.5–2.0 μM indole-3-butyric acid. Regenerants were transferred to vermiculite and successfully established under an ex vitro environment in garden soil.     

Flow cytometry and start codon targeted (SCoT) genetic fidelity assessment of regenerated plantlets in Tylophora indica (Burm.f.) Merrill

Tylophora indica (Burm.f.) Merrill. is a pharmacologically important plant, popular for alkaloidal and non-alkaloidal richness. Large scale propagation of T. indica is difficult in the wild as the seeds are small and the frequency of germination is very poor. In the present study, the genome size estimation of in vitro regenerated (indirect, direct and somatic embryo mediated) T. indica was made by flow cytometric method. Clonal fidelity of the regenerants was assessed using a start codon targeted (SCoT) molecular marker. Initially, the explants were inoculated on Murashige and Skoog basal medium supplemented with various concentrations of plant growth regulators like 2,4-dichlorophenoxy acetic acid (2,4-D), Kinetin, 6-benzyl amino purine (BAP) and 1-naphthalene acetic acid either singly or in combinations. The highest callus induction frequency (87.75%) was obtained in 6.7 µM 2,4-D added MS medium which metamorphosed into progressive stages (globular, heart, torpedo, and cotyledonary) of embryos. Mature and healthy somatic embryos efficiently germinated into plantlets on 8.8 µM BAP?+?1.4 µM GA₃ enriched MS medium. Histological and scanning electron microscopic study confirmed the above developing stages. The regenerated shoots were rooted best in 2.45 µM Indole-3-butyric acid supplemented solid MS medium. The plants were hardened and acclimatized with 90% survivability. The flow cytometric 2C DNA content of indirect, direct and somatic embryo derived plants was 1.896 pg, 1.940 pg and 1.926 pg respectively, very similar to the mother plant (1.928 pg). SCoT marker generated a high percentage of monomorphic bands (94%) revealing similarity with the mother plant, thus ensuring genetic fidelity. To the best of our knowledge, this is perhaps the first ever report of 2C DNA content estimation and SCoT marker based genetic homogeneity study in T. indica.

     

Plant regeneration in Spanish cedar, <Emphasis Type="Italic">Cedrela odorata</Emphasis> L., using zygotic embryo explants from mature seed and improvement of embryogenic nodule initiation by heat shock

Conditions for induction of embryogenic nodules and subsequent somatic embryogenesis in the tropical hardwood Cedrela odorata are described. Embryo explants from ungerminated mature seeds were placed on Driver and Kuniyuki Walnut (DKW) medium using 0.8% w/v agar as the gelling agent, plus benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D). Various phytohormone combinations were tested, from which 5 μM BA + 50 μM 2,4-D were chosen as the standard regime based on a maximum frequency of embryogenic nodule occurrence of 20–25% on this medium. Nodules, when excised from the cotyledons and placed on growth-regulator-free medium, produced both plantlets and secondary embryogenic tissue. With extended culture on growth-regulator-free DKW medium, plantlets developed roots and could be transplanted into pots for further growth. The frequency of nodule initiation could be improved by either orienting the cotyledon explants with their lower (abaxial) surface in contact with the medium or imposing a pre-excision period of heat shock. The treatments together were additive. An optimum heat-shock temperature (47°C) and range of exposure times (8–12 h) were defined.     

Development of an embryogenic callus induction method for centipede grass (<Emphasis Type="Italic">Eremochloa ophiuroides</Emphasis> Munro) and subsequent plant regeneration

The present study demonstrates the establishment of embryogenic tissue from seeds and (seedling-derived hypocotyls) shoot base explants derived from seedlings of Eremochloa ophiuroides. The highest percentage of callus induction obtained from seed and young shoot base explants was 52.0% and 66.6% on Murashige and Skoog (MS) basal media supplemented with 9.0 μM and 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. The type of callus obtained from both types of explants was off-white to yellow in color and non-friable and shiny in texture. Excised callus from the explants was subcultured onto fresh media of the same recipe for further proliferation. After 10–12 d of subculture, a yellow, globular, friable embryogenic callus was obtained from the initial callus. The highest percentage of embryogenic calli obtained at 40.0% was observed on media containing 2.2 μM 2,4-D. The highest regeneration rate of 46.6% was observed on MS media supplemented with 0.4 μM 2,4-D and 2.2 μM benzylaminopurine (BA). Regenerated shoots were rooted in MS basal medium. Plants with well-developed roots were transferred to pots containing a soil mix and acclimatized in greenhouse conditions. Four weeks post-transfer, acclimatized plants showed 100% survival and remained healthy and green. This is the first report of a successful method for induction of somatic embryogenesis with subsequent plant regeneration in centipede grass and demonstrates the establishment of embryogenic callus and efficient plant regeneration with potential application in the development of genetic transformation systems for centipede grass.     

Somatic embryogenesis in Agave tequilana Weber cultivar azul

Somatic embryogenesis was achieved from leaves of Agave tequilana Weber cultivar azul utilizing MS medium supplemented with L2 vitamins and the addition of cytokinins: 6-benzylaminopurine (BA), 1-phenyl-3(1,2,3-thiadiazol-5-yl)urea (TDZ), 6-(γ-γ-dimethylamino)purine (2ip) and 6-furfurylaminopurine (KIN), combined with the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Differences among the six genotypes studied with regard to their embryogenic response in culture were found. Embryos produced by genotype S3 under a hormone regime of high cytokinin (44.4 to 66.6 μM BA) compared to auxin (4.5 μM 2,4-D) contained chlorophyll, whereas those produced when auxin was high compared to cytokinin (9.0 and 13.6 μM 2,4-D and 1.3 and 4.0 μM BA, respectively) were whitish and morphologically similar to their zygotic counterparts. Somatic embryos matured and germinated after transferring the embryogenic calli to maturation and germination medium without growth regulators and enriched with organic nitrogen. Microscopic observations demonstrated a unicellular origin for production of indirect somatic embryos.     

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.     

Regeneration of Panax ginseng C.A. Meyer by organogenesis and nuclear DNA analysis of regenerants

Plant regeneration ability of ginseng (t Panax ginseng C.A. Meyer) via organogenesis was studied. Compact callus was induced from four different types of explants-leaf, petiole, flower stalk, and root of t in vitro-grown plantlets. Petioles were found to be the best material for callus induction. Calli induced on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (4.5 μM) and kinetin (0.46 μM) were conditioned for two weeks on the same medium. These calli differentiated into adventitious shoots when cultured on 1/2MS basal medium plus kinetin 4.7 μM and silver thiosulphate 10 μM. An addition of GA₃ (2.9 μM) and BA (4.4 μM) to MS basal medium, however, induced high frequency t in vitro flowering (86.1%) and multiple shoot budding which affected the normal complete development of plantlets. Plantlets with a well-developed root system were obtained six weeks after regenerated shoots had been transplanted to 1/2 MS20 medium containing IBA 1.2 μM. Nuclear DNA content was measured to check the stability of their ploidy level. Based on DNA flow cytometric analysis, all the regenerants were typically diploids as the mother plants were, indicating that nuclear DNA content remained stable during cell differentiation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth and net photosynthetic rates of Eucalyptus tereticornis Smith under photomixotrophic and various photoautotrophic micropropagation conditions

Calli were induced from leaf explants of seedling in Citrus grandis (L.) Osbeck (pummelo) on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA), 2,4,5-trichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic, 4-chlorophenoxyacetic acid, 4-methoxy-3,6-dichlorobenzoic acid or 4-amino-3,5,6-trichloropicolinic acid. 2,4-D was most effective. Only green, compact calli induced by 2,4-D at low concentrations (0.9 and 4.5 M) were capable of shoot formation and regenerated more than 13 shoots per callus on MS medium containing at least 6.66 M benzyladenine (BA). Calli induced by other auxins did not regenerate shoots on MS medium containing BA at all concentrations studied. A multiplication rate of 5–7 shoots was achieved from shoot tip culture on MS medium with 0.89 M BA. Roots developed when regenerated shoots were cultured on MS medium with 9.84 M indole-3-butyric acid and 5.37 M NAA. No response was obtained on mature leaves cultured on MS medium supplemented with the above mentioned auxins at various concentrations.