Plant regeneration via somatic embryogenesis from solid and suspension cultures of Vitis vinifera L. cv. ‘Manicure Finger’

Vitis vinifera L. cv. ‘Manicure Finger’ is one of the major table grape varieties in China. To provide a strong foundation for genetic transformation with potential for crop improvement, we undertook plant regeneration via somatic embryogenesis. Anthers and gynoecia were harvested from immature flowers and used as explants to induce embryogenic calli. Explants cultured in MS1 medium (based on Murashige and Skoog basal salts), supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4-μM 6-benzylaminopurine (6-BA) showed the highest rates of embryogenic callus induction (3.7%?±?1.3% for anthers and 4.8%?±?2.5% for gynoecia). After several months, somatic embryos were produced from embryogenic calli cultured in plant growth regulator-free MS2 medium (with reduced sucrose). Somatic embryos (SE) at the cotyledonary stage were isolated and cultured on three different media (MS2, MS3, or B) for conversion into plantlets, the efficiency of which ranged from 63.9%?±?4.8% to 83.9%?±?8.4%. After 1 mo of in vitro culture, 80% of plants with at least six leaves were successfully transplanted into soil. SE was repeatedly induced from previously induced somatic embryos for up to 1.5 yr. Using embryogenic calli as starting material, suspension cultures containing embryogenic cell aggregates were also established in liquid MS medium supplemented with 4.5-μM 2,4-D. The embryogenic cell aggregates continued to proliferate without differentiating for successive subculture cycles. After transfer to 2,4-D-free liquid medium for 4 wk, an average of 63.7%?±?9.0% mature SEs were produced per 20 mL of liquid medium. More than 40% of somatic embryos at cotyledonary stage, derived from the suspension cultures, successfully germinated into plants using solid medium.     

Direct somatic embryogenesis from leaf and petiole explants of Spathiphyllum ‘Supreme’ and analysis of regenerants using flow cytometry

This study established a method of regenerating Spathiphyllum ??Supreme?? through direct somatic embryogenesis. Somatic embryos occurred in leaf and petiole explants cultured in the dark on a Murashige and Skoog basal medium supplemented with 2.27, 4.54, or 9.08???M N-phenyl-N??-1,2,3-thiadiazol-5-ylurea (TDZ) in combination with 1.08???M ??-naphthalene acetic acid or 2.26???M 2,4-dichlorophenoxyacetic acid (2,4-D). Explants with somatic embryos were transferred to fresh medium containing the same concentrations of growth regulators under lighted conditions for embryo conversion. The highest frequencies of leaf explants with somatic embryos and embryo conversion were both 84.4?%, which were induced by 9.08???M TDZ with 2.26???M 2,4-D. The frequencies for somatic embryo induction and embryo conversion were both 100?% when petiole explants were induced by 4.54???M TDZ with 2.26???M 2,4-D. The number of plantlets produced per leaf explant and petiole explant were as high as 67.4 and 74.4, respectively. Plantlets after transplanting to a soilless substrate grew vigorously in a shaded greenhouse. Liners were stable without phenotypic variation. Flow cytometry analysis of randomly selected plants showed that they all had a single identical peak. The mean nuclear DNA index for ??Supreme?? was 1.568, and the nuclear DNA content was 14.222?pg 2C^?1. The estimated genome size for ??Supreme?? was 6,954.5?Mbp 1C^?1 with a CV at 4.008?%. The results suggest that the regenerated plants have a stable ploidy level and this established regeneration method can be used for highly effective propagation of uniform Spathiphyllum ??Supreme??.     

Regeneration of Syngonium podophyllum ‘Variegatum’ through direct somatic embryogenesis

A simple and effective method of regenerating Syngonium podophyllum ‘Variegatum’ via direct somatic embryogenesis has been established. Leaf and petiole explants were cultured on Murashige and Skoog (MS) medium supplemented with N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with either α-naphthalene acetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos directly formed at one or two sides of petiole explants on MS medium supplemented 2.5 mg l⁻¹ TDZ with 0.5 mg l⁻¹ NAA or 2.0 mg l⁻¹ TDZ with 0.2 mg l⁻¹ NAA or with 0.2 and 0.5 mg l⁻¹ 2,4-D, respectively. The frequency of petiole explants with somatic embryos produced was as high as 86% when cultured on medium containing 2.5 mg l⁻¹ TDZ with 0.5 mg l⁻¹ NAA. Up to 85% of somatic embryos were able to germinate after transferring onto medium containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ NAA. Approximately 50–150 plantlets were regenerated from a single petiole explant. However, there was no somatic embryo formation from leaf explants regardless of growth regulator combinations used. Regenerated plantlets from petiole explants were stable and grew vigorously after transplanting to a soilless container substrate in a shaded greenhouse.     

Efficient somatic embryogenesis and Agrobacterium-mediated transformation of pothos (Epipremnum aureum) ‘Jade’

Leaf and petiole explants of monocotyledonous pothos (Epipremnum aureum) ‘Jade’ were cultured on Murashige and Skoog basal medium supplemented with N-(2-chloro-4-pyridl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA). Somatic embryos appeared directly from explants after 4–8 weeks of culture; 9.1 μM TDZ with 1.1 μM NAA induced 61.1 % leaf discs and 94.4 % of petiole segments to produce plantlets through embryo conversion. Using this established regeneration method and an enhanced green fluorescent protein (GFP) gene (egfp) as a reporter marker, an Agrobacterium-mediated transformation procedure was developed. Leaf discs and petiole segments were inoculated with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pLC902 that contains novel bi-directional duplex promoters driving the egfp gene and hygromycin phosphotransferase gene (hpt), respectively. The explants were co-cultivated with strain EHA105 for 3, 5, and 7 days, respectively prior to selective culture with 25 mg l^?1 hygromycin. A 5-day co-cultivation led to 100 % of leaf discs to show transient GFP expression and 23.8 % of the discs to produce stable GFP-expressing somatic embryos. A 7-day co-cultivation of petiole explants resulted in the corresponding responses at 100 and 14.3 %, respectively. A total of 237 transgenic plants were obtained, and GFP fluorescence was observed in all plant organs. Regular PCR and quantitative real-time PCR analyses confirmed the presence of 1 or 2 copies of the egfp gene in analyzed plants. The highly efficient regeneration and transformation systems established in this study may enable genetic improvement of this vegetatively propagated species through biotechnological means.     

Primary and secondary somatic embryogenesis in Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’ and assessment of ploidy stability of somatic embryogenesis process by flow cytometry

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from petal explant of Chrysanthemum (Chrysanthemum morifolium) cv. ‘Baeksun’. Somatic embryogenesis was induced from petal explants on the Murashige and Skoog (MS) medium supplemented with 1.0 mg l^?1 2,4-dichlorophenoxyacetic acid (2,4-D) and 3.0 mg l^?1 6-benzyladenine (BA), yielding the highest mean number of embryos (56.3) per explant after 5 weeks of culture. We evaluated the effects of basal medium and various concentrations of sucrose on the proliferation of secondary somatic embryos. MS medium was observed to be more effective in promoting the proliferation of somatic embryos than half-strength Murashige and Skoog (1/2MS). In addition, 1 % sucrose was also found to be the best in induction of secondary embryogenesis. The highest germination rate (70 %) of the somatic embryos was observed on the MS medium containing 0.2 mg l^?1 α-naphthalene acetic acid and 1 g l^?1 activated charcoal (AC). Shoots elongated rapidly and roots developed well on hormone-free MS medium with 1 g l^?1 AC and successfully acclimated in the greenhouse. Flow cytometric analysis of the primary somatic embryos, secondary somatic embryos, and the somatic embryo-obtained plants along with the parent grown in the greenhouse showed that they all had same identical peaks, indicating that there was no variation of ploidy level during the regeneration process. We expect that our report would be useful for micropropagation and Agrobacterium-mediated genetic transformation studies of this cultivar.     

Shoot regeneration from petioles and leaves of Vitis X labruscana ‘Catawba’

Summary Shoot regeneration and normal plants were obtained from leaf and petiole explants derived from in vitro grown shoots of Vitis X labruscana Catawba. Regeneration was induced in the presence of both 6-benzylaminopurine and indole-3-butyric acid; combinations of 2,4-dichlorophenoxyacetic acid or 2-naphthoxyacetic acid with 6-benzylaminopurine did not permit regeneration from leaf explants. Up to 15% of leaf and 70% of petiole explants regenerated shoots on media with 5.0–10.0 M BA and 0.1–0.5 M IBA. Incubation in the dark was required to obtain regeneration. About 50% of shoots developed normally following transfer to light. An average of one shoot regenerated from leaf explants and 3.3 shoots regenerated per petiole explant. Regeneration from petioles and leaves was always from the basipetal end. The interaction of 6-benzylaminopurine with indole-3-butyric acid was also examined.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NN69 Nitsch and Nitsch (1969) medium - NOA 2-naphthoxyacetic acid     

Adventitious shoot regeneration from ovaries of Hosta ‘Golden Scepter’

Summary The objective of this study was to evaluate the ability ofHosta Golden Scepter (GS) ovary explants to generate adventitious shootsin vitro. Ovaries were transversely cut into halves and transferred to petri dishes containingHosta initiation medium supplemented with naphthaleneacetic acid (NAA) at 2.5 μM and N⁶-benzyladenine (BA) at 10 μM. GS produced adventitious shoots from the ovary base via organogenesis. The number of adventitious shoots regenerated from callus increased linearly with repeated subculturing on Murashige and Skoog (MS) medium supplemented with 2.5 μM NAA and 10 μM BA. The number of multiple shoots developing from callus (15.8), shoot tip (8.4), leaf (6.7), and root (4.3) occurred on MS medium supplemented with 2.5 μM NAA and 20–30 μM BA. There were significant differences in the number of shoots regenerated from shoot tips and callus on MS medium with 50 and 100 mgmyo-inositol per l. Similarly, there were significant differences in the number of axillary shoots and adventitious shoots produced with 20 g/l sucrose treatment.     

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.     

Plant regeneration via somatic embryogenesis of Elymus sibiricus cv. ‘chuancao No. 2’

The mature seeds, mesocotyls, and young leaf tips of Elymus sibiricus L. cv. ‘chuancao No. 2’ were cultured on Murashige and Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-d) and 0.05 mg/L kinetin in the dark at 26°C, the calluses were produced. The rate of callus regeneration depended on the explants source and plant growth regulators. Plants regenerated from whitish-yellow-coloured compact nodular callus formed after subculturing for 8 weeks. Higher frequency (54%) of shoot differentiation was obtained from the embryo tissues of mature seed than from either mesocotyls (24%) or young leaf tip tissues (6%) when these calluses from different types of explants were cultured on plant regeneration medium containing half strength MS salts supplemented with 0.1 mg/L kinetin, 1.5 mg/L 2,4-D and 20 g/L sucrose. The green plants were rooted within 6 weeks in the root regeneration medium, and over 97% of these soil-established plants were obtained in the greenhouse when potted in a sand and peat mixture medium.     

In vitro organogenesis and somatic embryogenesis from punctured leaf of Populus nigra × P. maximowiczii

Various explant sources of Populus nigra × P. maximowiczii were used to examine the effects of growth hormones on morphogenesis in vitro. Initial experiments indicated that punctured leaves were superior to non-punctured ones for both callus growth and formation of shoots and roots on MS medium containing various types and concentrations of growth hormones. After 6 weeks in culture, an average of 178 shoots, 129 roots and 3.1 g fresh weight of callus were directly produced from the abaxial side of each punctured leaf. The best combinations of growth hormones for shoot, root and callus proliferation were 0.88 M BA plus 0.05 M 2,4-D, 0.44 M BA plus 2.69 M NAA and 0.44 M BA plus 2.26 M 2,4-D, respectively. Embryoids were also formed on callus derived from punctured leaves. The number of embryoids varied from 0 to 30 per punctured leaf. Adventitious shoots also developed simultaneously with the embryos. Embryoids were removed with a scalpel at the early developmental stages and placed on MS medium lacking growth regulators. Regenerated plantlets were transferred to pots containing vermiculite for normal growth in the greenhouse.     

Somatic embryogenesis and shoot regeneration from excised adventitious roots of the rootstock Rosa hybrida L. ‘Moneyway’

Plants were regenerated from excised adventitious roots of the rose rootstock Moneyway via a three step procedure: callus induction, induction of somatic embryos and shoot development. Callus was induced on excised roots after incubation for 4 weeks in the dark on SH-medium (Schenk and Hildebrandt) containing 50 M 2,4-dichlorophenoxyacetic acid. For embryo induction, calluses were transferred to hormone-free SH-medium and incubated for 8 weeks. The use of Gelrite instead of agar during callus induction stimulated somatic embryogenesis (up to 16% of the explants formed organized structures), whereas the presence of 6-benzylaminopurine in this phase inhibited subsequent regeneration. Yellow solid calluses with embryo-like cotyledons or primordia and friable calluses with embryos were selected, and upon incubation in the light shoots developed. Shoot development was faster and more frequent on solid callus than on friable callus (64% and 21% of the calluses finally formed one or more shoots, respectively). Eleven out of thirteen regenerants developed similarly to control shoots. Finally this regeneration method is compared with other systems for somatic embryogenesis and opportunities for the production of transgenic rose rootstocks and rose cultivare are discussed.Abbreviations BAP 6-benzylaminopurine - BM basal medium - BM+ enriched basal medium - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI-4,6 diamidino-2-phenylindole - FeEDDHA ferric ethylenediamine di(ohydroxyphenylacetate) - FeEDTA ferric ethylenediamine tetraacetate - IBA indole-3-butyric acid     

Somatic embryogenesis ofCyclamen persicum Mill. ‘Anneke’ from aseptic seedlings

Summary InCyclamen persicum Anneke, explants from the various vegetative organs of aseptic seedling formed embryoids. The optimal responses were recorded in Murashige and Skoog (MS) medium enriched with 5.0µM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.5µM kinetin and 3–6% sucrose. Embryogenesis was enhanced at higher temperature of 25–30°C. On the other hand, light inhibited embryogenesis. Histological and morphological studies confirmed that the embryoids were indeed somatic embryos.Abbreviations MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid     

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...     

High-yielding repetitive somatic embryogenesis and plant recovery in a selected tea clone, ‘TRI-2025’, by temporary immersion

 Methods for improving the efficiency of repetitive somatic embryogenesis and plant recovery from somatic embryos of clonal tea, TRI 2025 [Camellia sinensis (L.) O. Kuntze] were investigated by optimising the immersion frequencies of the explants using a modified temporary immersion system (TIS). The relative efficiencies of three conventional methods for multiplying embryos were compared with the temporary immersion method. The highest rate of multiplication of secondary embryos (24-fold) was achieved using the TIS. By controlling the immersion cycles, we achieved more consistent, synchronised multiplication and embryo development with a high level of plant recovery. A one-step computer-programmed immersion protocol based on a single, simple medium with no growth regulators was developed, enabling multiplication, maturation, germination and plant recovery within 17 weeks. Plantlets recovered through this method were hardy, with 2- to 5-cm-long shoots containing a minimum of 2–4 lush green leaves and a well-formed taproot. Callus formation, hyperhydricity and other developmental abnormalities were not observed at any stage in the process. Plantlets produced using this method were successfully acclimatised to glasshouse conditions. This protocol avoids culture transfers, and thus minimises the risk of contamination and reduces labour costs. This technique could have significant commercial implications in tea propagation as it has the potential for large-scale production with considerably reduced production costs. Received: 28 June 1999 / Accepted: 20 April 2000     

Transgenic plantlets of ‘Chancellor’ grapevine (Vitis sp.) from biolistic transformation of embryogenic cell suspensions

Transgenic plantlets of Chancellor grapevine (Vitis L. complex interspecific hybrid) were produced via biolistic transformation. Embryogenic cell suspensions were bombarded with 1 m tungsten particles coated with pBI426 which encodes a fusion peptide between -glucuronidase (GUS) and neomycin phosphotransferase II (NPTII). The fusion peptide is under the control of a double 35S Cauliflower Mosaic Virus promoter and a leader sequence from Alfalfa Mosaic Virus. The cells were placed on kanamycin-containing media (10, 25 or 50 mg/l) 2 d after bombardment. Activated charcoal reduced cell browning. Embryos were first observed on selective media 14–29 weeks after bombardment. More than 1600 clusters of embryos were germinated and/or assayed for GUS. Of 621 embryos assayed for GUS expression, 182 (29.3%) were positive. PCR confirmed the presence of the NPTII gene in all 5 GUS-positive and 2 GUS-negative (bombarded) embryos tested. In germination experiments, 15% of the embryo clusters produced at least one plant with normal shoot growth. Of 164 normal plants assayed for GUS expression, 37 (22.6%) were positive. The NPTII gene was amplified by PCR in 1 (of 1) GUS-positive and 4 (of 5) GUS-negative bombarded plants, but not in non-bombarded control plants. Southern blotting confirmed integration of the NPTII gene in all 3 of the GUS and PCR-NPTII positive plants tested. Biolistics is an efficient method for transformation of Chancellor and should be applicable to other important grape cultivars.Abbreviations AC activated charcoal - GUS -glucuronidase - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - NAA -naphthalene acetic acid - TDZ thidiazuron - NPTII neomycin phosphotransferase II - Km kanamycin - MS Murashige and Skoog (1962) medium - WPM Woody Plant Medium of Lloyd and McCown (1980)     

Segregation of genetic markers among plants regenerated from cultured anthers of broccoli (Brassica oleracea var. ‘italica’)

Summary An experiment was conducted to determine critical factors in the recovery of embryos from cultured anthers of broccoli (Brassica oleracea var. italica) and to unambiguously distinguish whether embryos were of gametophytic origin. Among factors tested, genotype, genotype x anther developmental stage, and method of anther culture had a distinct impact on embryo recovery, whereas length of anther exposure to the culture medium did not. However, extreme heterogeneity of embryo emergence within and among replications precluded statistical contrasts. Among 762 plants derived from embryos of four independent cultivars, only one was determined to be of sporophytic origin by use of heterozygous codominant isozyme markers. Two of the cultivars tested were heterozygous at two or more loci. While segregation among loci was consistent with previously published linkage data, segregation of alleles was consistently non-random. In all of seven separate cases involving four cultivars, a significant over-representation of the fast-migrating class was observed. It appears, therefore, that populations of plants derived from microspores within cultured anthers of broccoli do not necessarily represent a random gametic array, and that care must be exercised in breeding and genetic applications.     

Factors influencing secondary somatic embryogenesis inMalus x domestica Borkh. (cv ‘Gloster 69’)

A procedure for regeneration of apple plants through secondary somatic embryogenesis (SSE) was developed in apple Gloster 69. Primary somatic embryos were produced from cotyledon-derived cultures of immature zygotic embryos. These somatic embryos were multiplied by secondary somatic embryogenesis (SSE) on media with different Plant Growth Regulator (PGR) combinations. The highest SSE rate (55.5%) was obtained with a combination of NAA (5.3 M), BAP (0.9 M) and KIN (0.9 M) or with TDZ alone (10 M). In addition, effects of explant source, somatic embryo size, type and concentrations of carbohydrates and gelling agents on SSE were investigated. The optimum SSE (>73%) was obtained by the culture of large size somatic embryos or cotyledon-like structures on medium containing a combination of NAA/BAP/KIN or TDZ (10 M) alone, maltose (175 mM) and Phytagel (2.8 g/1).     

‘Hemiepiphyte’: a confusing term and its history

Background and Scope

Over more than 120 years of scientific study since Schimper''s seminal work, the recognized categories of structurally dependent plants have changed several times. Currently, ignoring parasitic mistletoes, it is usual to distinguish four functional groups: (1) true epiphytes; (2) primary hemiepiphytes; (3) secondary hemiepiphytes; and (4) climbing plants, i.e. lianas and vines. In this Viewpoint, it is argued that the term secondary hemiepiphytes (SHs) is misleading, that its definition is hard to impossible to apply in the field and, possibly causally related to this conceptual problem, that the use of this category in field studies is inconsistent, which now hampers interpretation and generalization.

Conclusions

Categories will frequently fail to capture gradual biological variation, but terms and concepts should be as unambiguous as possible to facilitate productive communication. A detailed analysis of the conceptual problems associated with the term SH and its application in scientific studies clearly shows that this goal is not fulfilled in this case. Consequently, the use of SH should be abandoned. An alternative scheme to categorize structurally dependent flora is suggested.