Plantlet regeneration from mature zygotic embryos and embryonic explants of masson pine （Pinus massoniana Lamb.）

Excised zygotic embryos,cotyledons and hypocotyls of juvenile seedlings of masson pine were grown on DCR medium supplemented with several concentrations of various plant phytohormones.BA(1.0mg/L) in combination with NAA(0.05mg/L) in DCR medium was found to increase the formation of adventitious buds from mature zygotic embryos,but most of them were formed at the tips of embryonic cotyledons.Adventitious buds were obtained from cotyledons and hypocotyls from juvenile seedlings when they were cultured on DCR medium containing BA 3-5 mg/L and NAA 0.1-0.2 mg/L.Elongation of buds were observed on hormone-free DCR medium with or without activated charcoal(0.5%).Root initiation was achieved with full or half strength DCR medium supplemented with IBA 1.0 mg/L and NAA 0.25-0.5 mg/L.Approximately 11-20 axillary buds formed on each explant when juvenile seedling explants were treated(3-20h) with BA 50-100 mg/L,followed by transfer to hormone-free DCR medium.The maximum number of shoots obtained per explant within six months was 33.     

Callus induction and plant regeneration from different explant types of Miscanthus x ogiformis Honda ‘Giganteus’

Different explants of Miscanthus x ogiformis Honda Giganteus were tested in order to develop an efficient tissue culture system. Shoot apices, leaf and root sections from in vitro-propagated plants, and leaf and immature inflorescence sections from 6-month-old greenhouse-grown plants were used. The explants were cultured on Murashige and Skoog medium supplemented with 4.5, 13.6, 22.6 or 31.7 M 2,4-dichlorophenoxyacetic acid. Three types of callus were formed but only one was embryogenic and regenerated plants. Callus induction and formation of embryogenic callus depended on the type and developmental stage of the explants. Shoot apices formed the highest percentage of embryogenic callus. There was a difference in the formation of embryogenic callus between leaf explants from in vitro-propagated shoots and greenhouse-grown plants. The best results were obtained from newly formed leaves of in vitro-propagated shoots and older leaves of greenhouse-grown plants. Immature inflorescences smaller than 2.5 cm produced a higher percentage of embryogenic callus than larger more mature inflorescences. Embryogenic callus derived from immature inflorescences had the highest regeneration capacity. Differences in 2,4-dichlorophenoxyacetic acid concentrations had no significant effect on callus induction, embryogenic callus formation and plant regeneration.Abbreviations MS Murashige & Skoog - 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzyladenine - NAA 1-naphthaleneacetic acid - PPFD photosynthetic photon flux density     

Efficient and rapid plant regeneration of oil palm zygotic embryos cv. ‘Tenera’ through somatic embryogenesis

An efficient and rapid plant regeneration system through somatic embryogenesis was developed using 13-week-old zygotic embryos of oil palm (Elaeis guineensis Jacq.) cv. ‘Tenera’. Zygotic embryos were cultured on MS and N6 media supplemented with 2.0 mg L⁻¹ picloram, 2,4-D and dicamba. The highest embryogenic callus formation (32%) was observed on N6 medium with 2,4-D after 3 month culture on callus induction medium. Somatic embryos were continuously formed from nodular calli on embryo maturation medium [N6 + 0.1 mg L⁻¹ 2,4-D, 0.16 g L⁻¹ putrescine, 0.5 g L⁻¹ casein amino acids and 2.0 g L⁻¹ activated charcoal(AC)] for 3–5 months. Histological analysis confirmed that embryo development occurred via somatic embryogenesis. For plant regeneration, modified N6 medium (MN6) with AC (0.5 g L⁻¹) without growth regulators, induced both shoot and root formation simultaneously with the highest regeneration rate of 56%. This combined shoot and root induction protocol shortened the culture time to 9–12 months. Furthermore, after acclimatization, more than 85% of transferred plants from our protocol developed successfully in the soil.     

Somatic embryogenesis and plant regeneration from leaf-derived cell suspension of a mature tree — Thevetia peruviana L.

Cell suspension cultures, which retained embryogenic potential for almost 2 years, were established from young, expanding, juvenile leaves of a mature Thevetia peruviana L. tree. Calli were obtained by culturing young leaf discs on MS medium supplemented with 2 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.1 mg/L kinetin. Suspension cultures were initiated by transfer of calli to liquid medium containing 1 mg/L 2,4-D + 0.1 mg/L kinetin, and the cultures were maintained by subculturing to fresh medium at 2 week intervals. Embryogenic frequency of cell aggregates was more than 80% when plated on semi-solid medium containing 0.1 mg/L 2, 4-D and 2 mg/L 2-isopentenyladenine (2-iP). Cell aggregates with developing embryos were transferred to fresh medium lacking growth regulators for embryo maturation. Early embryo development was synchronous and a large number of somatic embryos were produced. These somatic embryos developed into plantlets upon subsequent transfer to modified half-strength MS medium. More than 200 green and rooted plants, at an average of 80 plants per 100 mg of embryogenic callus, were obtained with 60% survival under glass house conditions.Abbreviations 2, 4-D 2 4-dichlorophenoxyacetic acid - 2-iP 2-isopentenyladenine - IAA Indole — 3 — acetic acid - KN Kinetin - MS Murashige and Skoog (1962) basal medium - NAA 1 -Napthalene acetic acid     

Electroporation-mediated improvement of plant regeneration from colt cherry (Prunus avium × pseudocerasus) protoplasts

Results are presented that show a promotory carry-over effect, of an electroporation treatment of isolated cell suspension protoplasts of Colt cherry (Prunus avium × pseudocerasus), on the growth of protoplast-derived calli and on plant regeneration capacity. Callus from protoplasts subjected to three successive exponential pulses at 250 V or 500 V showed the largest fresh weight increases between subcultures, and also exhibited the highest frequency of plant regeneration based on the number of shoots per callus. These shoots, in turn, produced a more prolific root system when compared to those derived from non-electropulsed protoplasts.     

Callus formation and plant regeneration through direct culture of isolated pollen of Hordeum vulgare cv. ‘Sabarlis’

Summary Pollen calli and plantlets of Hordeum vulgare cv. Sabarlis were obtained through direct pollen culture without pretreatment of spikes or preculture of anthers. Isolated immature pollen grains were cultured first in a 0.3 M mannitol solution or a C1 basal medium (Chen et al. 1979) supplemented with 0.3 M mannitol but without sucrose for 5–7 days, then transferred into a C1 medium containing 6% sucrose, 3 mM glutamine and 5 mM m-inositol. After a 3 week culture period small pollen calli derived from the pollen grains were transferred into a growth medium comprising C1 basal medium supplemented with 250 mg/1 lactalbumin hydrolysate and 0.5 mg/1 kinetin. For shoot regeneration, vigorously growing calli were transferred onto agarsolidified MS medium (Murashige and Skoog 1962) containing 3% sucrose, 2 mg/1 benzyladenine and 0.5 mg/1 indole-3-acetic acid. The ratio of green plants to albino was approximately 12.2.     

Effect of light conditions on anatomical and biochemical aspects of somatic and zygotic embryos of hybrid larch (Larix?×?marschlinsii)

Background and Aims In conifers, mature somatic embryos and zygotic embryos appear to resemble one another physiologically and morphologically. However, phenotypes of cloned conifer embryos can be strongly influenced by a number of in vitro factors and in some instances clonal variation can exceed that found in nature. This study examines whether zygotic embryos that develop within light-opaque cones differ from somatic embryos developing in dark/light conditions in vitro. Embryogenesis in larch is well understood both in situ and in vitro and thus provides a suitable system for addressing this question.Methods Features of somatic and zygotic embryos of hybrid larch, Larix × marschlinsii, were quantified, including cotyledon numbers, protein concentration and phenol chemistry. Somatic embryos were placed either in light or darkness for the entire maturation period. Embryos at different developmental stages were embedded and sectioned for histological analysis.Key Results Light, and to a lesser degree abscisic acid (ABA), influenced accumulation of protein and phenolic compounds in somatic and zygotic embryos. Dark-grown mature somatic embryos had more protein (91·77 ± 11·26 µg protein mg^–1 f.wt) than either dark-grown zygotic embryos (62·40 ± 5·58) or light-grown somatic embryos (58·15 ± 10·02). Zygotic embryos never accumulated phenolic compounds at any stage, whereas somatic embryos stored phenolic compounds in the embryonal root caps and suspensors. Light induced the production of quercetrin (261·13 ± 9·2 µg g^–1 d.wt) in somatic embryos. Mature zygotic embryos that were removed from seeds and placed on medium in light rapidly accumulated phenolics in the embryonal root cap and hypocotyl. Delaying germination with ABA delayed phenolic compound accumulation, restricting it to the embryonal root cap.Conclusions In larch embryos, light has a negative effect on protein accumulation, but a positive effect on phenol accumulation. Light did not affect morphogenesis, e.g. cotyledon number. Somatic embryos produced different amounts of phenolics, such as quercetrin, depending on light conditions. The greatest difference was seen in the embryonal root cap in all embryo types and conditions.     

Isolation,culture and plant regeneration of colt cherry (Prunus avium × pseudocerasus) protoplasts

Large numbers of viable protoplasts were isolated from leaf mesophyll tissues and cell suspension cultures of Colt cherry using 1% (w/v) Onozuka R-10, 0.2% (w/v) Macerozyme R-10, 0.1% (w/v) Driselase, 1% (w/v) polyvinylpirrolidone (av. MW 10 000) (PVP-10) and 2% (w/v) Meicelase, 2% (w/v) Rhozyme HP-150 and 0.03% (w/v) Macerozyme R-10. Culture media, based on Murashige and Skoog's (MS) salts, supplemented with 9% (w/v) mannitol and various combinations of α-naphthalene acetic acid (NAA), 6-benzylaminopurine (BAP) and zeatin (Z) promoted cell wall regeneration followed by cell colony and callus formation. Protoplasts of both sources were compared in relation to their cultural requirements. Protoplast-derived callus underwent organogenesis.     

Induction of multiple shoots and plant regeneration from ‘accessory buds’ of nodal segments from field-grown mature cotton plants (Gossypium hirsutum L.)

Summary Sprouting of a single shoot was obtained from nodal segments of field-grown cotton plants (Gossypium hirsutum L. cv. DCH-32 and NHH-44) when cultured on Murashige and Skoog's (MS) basal medium devoid of plant growth regulators. Pruning of the sprouted shoot followed by re-culturing of the explant on MS basal medium supplemented with 2.22 μM benzylaminopurine triggered the dormant ‘accessory buds’ present in the node to proliferate and differentiate into multiple shoots. The shoots elongated on the same medium and rooted on MS basal medium devoid of growth regulators. Plantlets were hardened under greenhouse conditions and transferred to the field. Flowering and boll formation were observed in these plants at maturity. This technique for successful clonal propagation from mature cotton tissues should permit the rapid multiplication of plants selected based on specific phenotypic or genotypic characteristics.     

A new species of Alstroemeria (Alstroemeriaceae) from Pará, Brazil

A new species,Alstroemeria paraensis, from Pará, Brazil, is described and illustrated. This species is characterized by its robust floral stem, reduced leaves, congested inflorescence, and maculated inner and outer tepals.     

Long-term effect of electroporation on enhancement of growth and plant regeneration of colt cherry (Prunus avium × pseudocerasus) protoplasts

Electric pulses applied to Colt cherry protoplasts enhanced the long-term growth and plant regeneration of protoplast-derived tissues. Protoplasts isolated from long-term cultured tissues derived from electroporated protoplasts retained the ability to enter division in culture earlier and with a higher frequency of plant regeneration than untreated cell suspension protoplasts.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - IBA 4-indole-3yl-butyric acid - MES 2-N-morpholinoethane sulphonic acid - MS Murashige and Skoog (1962) - NAA -naphthaleneacetic acid - PE plating efficiency - Z zeatin     

Recovery of somatic embryos and plantlets from protoplast cultures of Larix × eurolepis

Summary Somatic embryos and plantlets were regenerated from protoplasts of hybrid larch (Larix × eurolepis) isolated from two embryogenic callus and cell suspension culture lines (L1 and L2). L2, which was highly embryogenic, consistently yielded protoplasts that gave rise to somatic embryos. Centrifugation on a discontinuous medium/Percoll density gradient resulted in accumulation of embryogenic protoplasts in one of the Percoll interfaces. First division frequencies were in the range of 28–39% in line 1 and 18–20% in line 2 in both liquid and agarose-solidified culture media. The critical factor in maintaining high viability of cultures was lowering of osmotic pressure by dilution of the initial medium. The first somatic embryos were detected in 23- to 28-day-old cultures. Some of these developed into plants that were transferred to soil.     

Plant regeneration from seedling explants of Eucalyptus grandis × E. urophylla

Hypocotyls, cotyledons, cotyledonary nodes and primary leaves were used as explants to establish a regeneration protocol for Eucalyptus grandis × E. urophylla. These seedling-derived explants were incubated on a modified MS medium (SP medium), supplemented with 2.0 M TDZ. After 1 month, the calluses obtained were transferred to SP medium containing different concentrations of BA and NAA or zeatin and NAA. Shoots were induced from these calluses at a high frequency. Shoot elongation was then stimulated on SP medium supplemented with BA, NAA and GA₃ for 20 to 30 days. For rooting, 50 mm long shoots were cultivated on root induction medium containing IBA (2.5 M) for different periods and then transferred to the same medium but without auxin, for 30 days. Plantlets were then successfully transplanted to greenhouse conditions.     

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

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

High-efficiency direct somatic embryogenesis and plant regeneration from leaf base explants of “peperina” (Minthostachys verticillata)

In Vitro Cellular & Developmental Biology - Plant - In vitro culture has been recognized as a potential plant clonal propagation tool for a broad variety of commercial, ornamental, and...     

Callus culture and an unconventional pattern of sporophyte regeneration in Drynaria quercifolia—A medicinal fern

Summary Callus induction and regeneration studies were carried out on a medicinal fern, Drynaria quercifolia native to Asian countries. It is a seasonal fern that regenerates only during the monsoons. Callus was induced on Knop’s (1865) medium supplemented with 20 gl⁻¹ sucrose, 8gl⁻¹ agar, and either 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), or indole-3-butyric acid at different concentrations. Morphogenetic callus obtained on 5 mgl⁻¹ 2,4,5-T was subcultured onto solid and liquid media (shaken flask and discontinuously stirred bioreactor cultures) for callus proliferation and regeneration studies. A significant amount of sporophyte regeneration was observed on solid medium containing 10 mgl⁻¹ 6-(δ, δ-dimethylallylamino) purine (2iP). Sporophyte regeneration from callus followed an atypical pattern of development. Leafy structures of single-cell thickness with a microrhizome were formed as sporophyte initials. Prolonged cultures of these structures resulted in the formation of juvenile sporophytes in vitro. The use of liquid media resulted in increased biomass in culture. The present study is the first report of a successful system for callus production and regeneration of sporophytes from leafy structures in ferns. The method can be successfully applied for generation of biomass of D. quercifolia, throughout the year.     

Phenotypic variation and ploidy level of plants regenerated from protoplasts of tetraploid potato (Solanum tuberosum L. cv. ‘Bintje’)

Summary A wide range of phenotypic variation occurred among protoplast — derived plants of tetraploid potato cultivar Bintje. The variant plants had alterations in growth and vigour, and in leaf and stem characteristics. The results suggest that the altered morphologies are caused predominantly by changes in ploidy levels. Some alterations could be attributed typically to octoploidy and aneuploidy. The occurrence of mixoploidy indicates that at least part of the observed variation arose during culture stage. The exogeneous cytokinin or auxin level and their combination during in vitro phase influenced the frequency of the variants observed. The origin of variation is discussed.     

The vascular plant collections of R. S. Williams from Bolivia and Peru (1901–1902)

The life and career of Robert Statham Williams (1859–1945) is sketched. His trip to Bolivia and Peru in 1901–1902 is discussed at length and an itinerary, with dates, is provided. Details concerning the numbering and distribution of his vascular plant collections are given, and notes are provided concerning the typification of the new species that Henry Hurd Rusby (1855–1940) based on these collections. An annotated catalog of vascular plant species described from Williams' Bolivian and Peruvian plant collections is appended, as is a gazetteer of the place names cited in Williams' field books and publications based on Williams' vascular and cryptogamic plant collections. One new combination,Oxypetalum apoloensis, and one new name,Acalypha rusbyi, are proposed. Lectotypes are designated forGothofreda apoloensis andCedrela brunellioides.