This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.

Using immature embryos and cotyledons as explants, a successful system to culture immature embryos and induce direct regeneration from cotyledons was established for Prunus mume “Xuemei”. For immature embryo culture, a high frequency of plantlet formation (89.5%) from the embryonic axis was obtained using half-strength Murashige and Skoog (1/2 MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). Shoots formed directly from cotyledons with the embryo axis intact when explants were cultured on 1/2 MS medium containing 2.2 μM BA with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when the embryonic axis was removed from the cotyledons and cultured on 1/2 MS medium supplement with 13.2 μM BA, 2.7 μM NAA or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA, respectively. Regenerated shoots were successfully rooted on 1/2 MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of the embryonic axis, BA, and TDZ on cotyledon regeneration was investigated in detail. Rooted plantlets were transferred to soil successfully.

This is the first report on somatic embryogenesis in common ash (Fraxinus excelsior L.). Experiments on somatic embryogenesis induction were carried out on zygotic embryos at different phases of development and maturation. The embryo axes were isolated and cultured on media containing different plant growth regulators (PGRs). Embryogenic tissues were obtained from embryos collected at an incomplete maturation phase and cultured on a modified Murashige and Skoog medium containing 8.8 μM 2, 4-dichlorophenoxyacetic acid and 4.4 μM benzyl-adenine (BA). Embryos isolated from seeds at an advanced stage of maturation showed only organogenetic phenomena. Embryogenic tissues were successfully subcultured and multiplied on medium containing a reduced concentration of PGRs. After their isolation, somatic embryos were induced to develop and mature by transfer to PGR-free medium and subsequent culture on medium containing 0.1 μM BA. Somatic embryos developed completely and also germinated spontaneously. Embryo germination and conversion were significantly improved when subjected to a period of storage at 4°C and transplant onto woody plant medium. Plantlets were successfully transferred to soil and acclimatized in a “misted” greenhouse.

We have evaluated the effects of the antibiotic hygromycin B on cotton (Gossypium hirsutum L.) callus induction, callus proliferation, and seed germination. Nontransgenic cotyledon and hypocotyl showed obvious variance in tolerance to hygromycin. Cotyledons were more sensitive to hygromycin than hypocotyls. Hygromycin at 7.5 and 20 mg l⁻¹ completely inhibited callus initiation from cotyledon and hypocotyl explants, respectively. Nontransformed calli did not grow on media supplemented with 10 mg l⁻¹ hygromycin and were killed at 15 mg l⁻¹. In seed germination assay, the presence of 20 mg l⁻¹ hygromycin significantly suppressed shoot and root elongation of seedlings. This hygromycin concentration was applied to select regenerated transgenic plantlets and their progenies. Based on these results, we developed an efficient hygromycin selection protocol for Agrobacterium-mediated cotton transformation and regeneration.

Studies on rooting of microshoots of smokebush (Cotinus coggygria Mill, var. Royal Purple), a woody ornamental, were carried out in vitro. Microshoots were rooted in a mixed-auxin regime (indole 3-acetic acid, indole butyric acid [IBA], and naphthalene acetic acid) or singly in the above auxins and the 2,4 dichlorophenoxyacetic acid (2,4-D) over a wide concentration range. Indole butyric acid at 10 μM proved to be the most suitable treatment, producing less basal callus, 100% rooting, and earlier root emergence than the other treatments. No roots were formed with 2,4-D. A 6-day root induction period was obtained with 10 μM of IBA. Histological studies revealed increased mitotic activity after 3 d in culture in the medullary ray cells, which led to root primordium formation, several of which were formed simultaneously around the base of the explant. The vascular tissues of these primordia connected to those of the explant, and roots began to emerge from the base by day 10. Thus, direct rhizogenesis occurred with the IBA treatment, as opposed to the roots that were formed in the basal callus under the mixed-auxin regime.

The factors affecting the induction and development of somatic embryos and plantlet acclimatization of peach palm (Bactris gasipaes Kunth) were evaluated to establish an efficient regenerative protocol based on somatic embryogenesis. Mature zygotic embryos were cultured in Murashige and Skoog (MS) medium supplemented with 0–40 μM of picloram (4-amino-3,5,6-trichloropicolinic acid) and 0 or 5 μM of 2-isopentyladenine (6-dimethylami-nopurine) (2-iP). After 5 mo. in culture embryogenic callus arose from primary calli. Picloram (10 μM) was effective in inducing embryogenic calli in 9.8% of the explants. The use of 1 μM of AgNO₃ enhanced embryogenic competence. Embryogenic calli showed an organized structure, a globular aspect, and were white to yellowish in color. Histological analyses showed that cell proliferation arose from subepidermal cells adjacent to vascular bundles, resulting in primary callus formed by a meristematic zone from which somatic embryos arose. Protein profile analyses revealed two high molecular mass bands in these embryogenic calli, but not in other tissues. Embryogenic calli were transferred to a culture medium containing 40 μM of 2,4-dichlorophenoxyacetic acid, 10 μM of 2-iP, plus 1 g l⁻¹ of glutamine, hydrolyzed 0.5 g l⁻¹ casein, and activated 1.5 g l⁻¹ of charcoal. Morphogenetic responses achieved in this medium were the development of somatic embryos, rooting, and loss of embryogenic capacity. Somatic embryos were converted to plantlets on MS medium plus 24.6 μM of 2-iP and 0.44 μM of naphthalene acetic acid. Plantlets were maintained in MS medium with activated charcoal (1.5 g l⁻¹) until they were 6 cm tall, and then acclimatized. After 16 wk, 84.2±6.4% survival was observed.

This study reports spore germination, early gametophyte development and change in the reproductive phase of Drynaria fortunei, a medicinal fern, in response to changes in pH and light spectra. Germination of D. fortunei spores occurred on a wide range of pH from 3.7 to 9.7. The highest germination (63.3%) occurred on ½ strength Murashige and Skoog basal medium supplemented with 2% sucrose at pH 7.7 under white light condition. Among the different light spectra tested, red, far-red, blue, and white light resulted in 71.3, 42.3, 52.7, and 71.0% spore germination, respectively. There were no morphological differences among gametophytes grown under white and blue light. Elongated or filamentous but multiseriate gametophytes developed under red light, whereas under far-red light gametophytes grew as uniseriate filaments consisting of mostly elongated cells. Different light spectra influenced development of antheridia and archegonia in the gametophytes. Gametophytes gave rise to new gametophytes and developed antheridia and archegonia after they were transferred to culture flasks. After these gametophytes were transferred to plastic tray cells with potting mix of tree fern trunk fiber mix (TFTF mix) and peatmoss the highest number of sporophytes was found. Sporophytes grown in pots developed rhizomes.

An efficient protocol was developed for in vitro clonal propagation of Curculigo orchioides Gaertn. through apical meristem culture. Multiple shoots were induced from apical meristems grown on Murashige and Skoog (MS) basal medium supplemented with 1.5 mg l⁻¹ 6-benzyladenine (BA), 100 mg l⁻¹ adenine sulfate (Ads) and 3% sucrose. Inclusion of indole-3-butyric acid (IBA) or indole-3-acetic acid (IAA) in the culture medium improved the formation of multiple shoots. The highest frequency of multiplication was obtained on MS medium supplemented with 1.5 mg l⁻¹ BA, 100 mg l⁻¹ Ads, 0.25 mg l⁻¹ IBA and 3% sucrose. Rooting was achieved upon transferring the micro-shoots to half-strength MS medium containing 0.25 mg l⁻¹ IBA and 2% sucrose. Micropropagtated plantlets were hardened in the greenhouse and successfully established in soil.

This study describes a reproducible protocol for rapid mass propagation of a multipurpose legume, Clitoria ternatea L., using cotyledonary node explants derived from axenic seedlings. Multiple shoots were induced in Murashige and Skoog (MS) medium supplemented with N ⁶-benzyladenine (BA), zeatin riboside, or thidiazuron. N ⁶-Benzyladenine at 1.0 mg l⁻¹ (4.44 μM) was most effective for shoot proliferation. Multiple shoots were also induced in nodal segments of in vitro-raised shoots grown on MS medium containing 1.0 mg l⁻¹ (4.44 μM) of BA. Rooting was best induced in shoots grown on half-strength MS medium with 0.25 mg l⁻¹ (1.42 μM) of indole-3-butyric acid. Plants were acclimatized in vermicompost and established in soil where they flowered and formed mature seeds.

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.

A three-stage procedure for embryogenesis in Trachyspermum ammi was developed from cotyledon and cotyledonary node explants cultured in Murashige and Skoog (MS) liquid medium supplemented with 0.2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Globular somatic embryos without intervening callus phase developed in 4 wk. The development of embryos to heart and torpedo stages required second-stage subculture of the explants (along with developing embryos) in liquid medium with lower concentrations of 2,4-D. Further development of embryos required a third-stage subculture in hormone-free liquid medium supplemented with 100 mg l⁻¹ casein hydrolysate. Regeneration of complete plantlets occurred after the fully developed somatic embryos were transferred to solidified half-strength MS medium supplemented with 1 mg l⁻¹ gibberellic acid.

This study describes a protocol for the induction of high frequency somatic embryogenesis directly from immature inflorescence explants in three sorghum genotypes (SPV-462, SPV-839, and M35-1). The effect of various growth regulators on somatic embryogenesis was investigated. High frequency somatic embrogenesis was obtained on Murashige and Skoog (MS) medium supplemented with 2 mg l⁻¹ 2, 4-dichlorophenoxyacetic acid (2, 4-D), and addition of 0.5 mg l⁻¹ kinetin (KN) in the medium further improved the formation of somatic embryos per explant in all genotypes. The presence of 1.5 mg l⁻¹ 6-benzylaminopurine plus 1.0 mg l⁻¹ KN in MS medium was most efficient for maturation and germination of somatic embryos. The genotype SPV-462 performed better than SPV-839 and M35-1 in terms of induction and germination of somatic embryos. Organogenesis also occurred in callus of all genotypes at the frequency of 20–25%. Regenerated plants from somatic embryos were successfully acclimatized in soil in the greenhouse where plants were grown to maturity, flowered, and set seeds. Regenerated plants appeared normal like that of the seed-raised plants.