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.     

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 from suspension cell cultures of Lilium×formolongi hort. Using a bioreactor system

Summary In vitro seedlings of Lilium × formolongi Hort. evs. Norikula, RaiZen No. 1, RaiZen No. 3, RaiZen Early, and Bailansa were used to induce callus by variously modified Murashige and Skoog (MS) media, using protocols for flask culture and bioreactor culture. Green embryogenic callus proliferated from roots near the base of bulblets of five varieties on media containing 0.53–5.3 μM α-naphthaleneacetic acid (NAA), and 28 cell lines were obtained by subcultures on the same medium. For flask culture, the fresh weight (FW) of embryogenic cell clumps doubled every 4 wk on MS basal salts supplemented with 0.53°M NAA and 30 g l⁻¹ sucrose. The maximum frequency of somatic embryos that developed into plantlets was 76.67±17% when plated onto solid MS basal medium without plant growth regulators (PGRs). Among the treatments using four types of bioreactors, the best cell growth and regeneration rate (74±0.14%) of somatic embryos was in a modified 2–1 bioreactor. Cells incubated in the other three bioreactors furned brown and died. Histological study revealed that regeneration was by somatic embryogenesis. The regenerants showed normal growth and flowering after 8–9 mo, in the field. A cell line of cv. Norikula has been subcultured in MS basal salts containing 0.53 μM NAA every 2 mo. for 6 yr. The cell aggregates became more synchronous and many typical embryogenic cells with dense cytoplasm were observed under a light microscope. The long-term embryogenic cells plated on MS basal medium still gave rise to numerous somatic embryos and converted into plantlets.     

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.     

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).     

Plant regeneration from calli of melon (Cucumis melo L., cv. ‘Amarillo Oro’)

Callus cultures from cotyledon and hypocotyl explants of a Spanish cultivar of melon (Amarillo Oro) have been tested for their growth and morphogenic capacity on a series of media with different concentrations of indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin). Melon tissues were able to undergo morphogenesis both via organogenesis and embryogenesis, depending on culture conditions and explant source. Shoot buds were obtained at high rates in cotyledon explants. In response to 1.5 mg/l IAA and 6.0 mg/l kinetin, more than 90% of the calli produced well-developed shoots. Hypocotyls failed to form shoots but formed somatic embryos on auxin containing media while cotyledon explants usually gave abundant shoots but only rarely formed embryos. It was possible to maintain organogenic callus lines for at least 12 months under defined conditions. Plants were recovered from adventitious shoots produced both in cotyledon-derived calli and from organogenic cell lines.     

Somatic embryogenesis in Rosa chinensis cv. ‘Old Blush’

Plant Cell, Tissue and Organ Culture (PCTOC) - Rose is one of the most widely used ornamental flowers in the world. However, the low induction rates for most rose somatic embryos means that it is...     

Agrobacterium mediated genetic transformation and plant regeneration via organogenesis and somatic embryogenesis from cotyledon leaves in eggplant (Solanum melongena L. cv. ‘Kecskeméti lila’)

Summary Novel and efficient protocols for plant regeneration and genetic transformation from longitudinally-halved cotyledons ofin vitro raised seedlings in eggplant (Solanum melongena L.) are described. After co-cultivation withAgrobacterium vectors harboring neomycin phosphotransferase (nptll) as selectable marker, transgenic plantlets were regenerated on selective media containing 100 mg/l kanamycin. Transformants were recovered from embryogenic calli induced by 4 mg/l-naphthaleneacetic acid (NAA), and from organogenic calli induced by the addition of 2 mg/l zeatin plus 0.01 mg/l NAA. Nineteen independent transgenic lines were grown to maturity. The structural integrity, expression and sexual transmission of the introduced genes for neomycin phosphotransferase and ß-glucuronidase (gus) were investigated.     

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.     

In vitro regeneration of Alstroemeria cv. ‘Yellow King’ by direct organogenesis

The common techniques for the in vitro production of Alstroemeria plants are based on rhizomes as explants, which have low multiplication rates and a high risk of carrying viral diseases. To overcome these problems, we developed a protocol for the in vitro regeneration of Alstroemeria cv.‘Yellow King’, by testing for shoot induction several explant sources (leaf, stem apices, rhizomes and immature inflorescence apices), temperature and light/dark regimes, hormone and salt concentrations. For shoot multiplication and rooting, several hormone concentrations were tested. We found that only the young floral apices produced adventitious shoots by direct organogenesis. The highest shoot induction rate (10.4 shoots per explant) was obtained by incubation in the dark for 15 days at 8 °C followed by 15 days at 25 °C and a 16-h/8-h light/dark regime, on a Murashige and Skoog (1962) liquid medium at 50% of the salt concentration, supplemented with 2.5 mg l⁻¹ KIN, 1.5 mg l⁻¹ BA and 1.0 mg l⁻¹ NAA, using a piece filter paper to support the explant. The highest shoot multiplication rate (9 shoots per explant) was obtained on a liquid MS medium at full strength supplemented only with BA at 1.0 mg l⁻¹. In vitro rooting of shoots was induced also on a liquid MS medium, either with or without plant hormones.     

Evaluation of haemoglobin (erythrogen): for improved somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L. cv. SVPR 2)

Somatic embryogenesis in cotton (Gossypium hirsutum L.) is accelerated when the plant regeneration medium is supplemented with haemoglobin (erythrogen). In cotton SVPR 2 lines, a higher frequency of embryoid formation was observed when the medium contained 400 mg/l haemoglobin. Fresh weight of the callus, rate of embryoid induction, number of embryoids formed and the percentage of plant regeneration from somatic embryos were increased. Among the two different cultivars tested, MCU 11 showed no response to the presence of haemoglobin when compared to SVPR 2, and embryogenic callus formation was completely absent in the former. Medium containing MS salts, 100 mg/l myo-inositol , 0.3 mg/l thiamine-HCL, 0.3 mg/l Picloram (PIC), 0.1 mg/l kinetin and 400 mg/l haemoglobin effected a better response with respect to embryogenic callus induction. After 8 weeks of culture, a high frequency of embryoid induction was observed on medium containing MS basal salts, 100 mg/l myo-inositol, 0.3 mg/l PIC , 0.1 mg/l isopentenyl adenine, 1.0 g/l NH₄NO₃ and 400 mg/l haemoglobin. Plant regeneration was observed in 75.8% of the mature somatic embryos, and whole plant regeneration was achieved within 6–7 months of culture. The regenerated plantlets were fertile and similar to in vivo-grown, seed-derived plants except that they were phenotypically smaller. A positive influence of haemoglobin was observed at concentrations up to 400 mg/l at all stages of somatic embryogenesis. The increase in the levels of antioxidant enzyme activities, for example superoxide dismutase and peroxidase, indicated the presence of excess oxygen uptake and the stressed condition of the plant tissues that arose from haemoglobin supplementation. This increased oxygen uptake and haemoglobin-mediated stress appeared to accelerate somatic embryogenesis in cotton.Abbreviations BAP Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - GR Glutathione reductase - 2iP Isopentenyl adenine - KT Kinetin - NAA Naphthaleneacetic acid - PFC Perfluorocarbon - PIC Picloram - PO Peroxidase - ROS Reactive oxygen species - SOD Superoxide dismutase - T.HCl Thiamine hydrochloride     

Micropropagation of Achillea filipendulina cv. ‘Parker’

Achillea filipendulina (family Asteraceae) is widespread throughout temperate North America. In order to clean stock plants from endemic fungal and bacterial contaminations a method for large-scale propagation of A. filipendulina through meristem culture was sought and found and is described in this paper. The best conditions for propagating A. filipendulina was found to be MS (Murashige and Skoog) salt medium supplemented with 3% sucrose and 1 mg l^–1 IAA (indole-3-acetic acid) plus 2 mg l^–1 BA (6-benzyladenine) under 16 h of cool fluorescent light. Rooted plants were successfully acclimatized within a short time after propagating on this medium. The propagation via tissue culture did not affect plant's presentation. The use of clean stock plants made it possible to increased Israeli production of Achillea from about 150,000 stems a year to about 1,300,000 stems a year.     

In vitro plantlet regeneration of Capsicum chinense Jacq. cv. ‘Bhut jalakia’: hottest chili of northeastern India

Chili (Capsicum chinense) cv. ‘Bhut jalakia’ is used in India for extraction of oleoresin and capsaicin as it is characterized by a very high capsaicin content. The conventional method of propagation of ‘Bhut jalakia’ is through seeds, but this is beset by short viability and low germination rates. Developing a suitable regeneration protocol for ‘Bhut jalakia’ was the focus of this study; as to date, in vitro regeneration for this cultivar has not been investigated. Cotyledon and shoot tip explants were cultured on Murashige and Skoog (MS) media supplemented with different concentrations of cytokinins and auxins. In the case of cotyledon explants, MS medium supplemented with 6-benzylaminopurine (BAP) at 35 μM and kinetin (KIN) at 15 μM were found to be optimal (4.00?±?0.57) for induction of multiple shoots per explant, whereas BAP at 14.8 μM and KIN at 60 μM were best (5.00?±?0.57) for growth of shoot tip explants. Shoots developed from cotyledon explants produced the maximum (8.67?±?0.32) number of roots on MS medium supplemented with low concentration (2.6 μM) of 2-naphthaleneacetic acid (NAA). Supplementation of indole-3-butyric acid (IBA) at 5 μM was found optimal for root formation (16.67?±?2.60) for shoots derived from of shoot tip explants. One month after transfer of in vitro regenerated plantlets to various potting mixes, the highest survival rate (40%) was observed in a mixture of sand, soil, and cow dung in a ratio of 1:1:1. Thus, both shoot tip and cotyledon explants may be cultured on MS medium modified with BAP, IBA, NAA, and KIN to regenerate ‘Bhut jalakia’ chili plants within 90 d.     

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.     

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.     

Micropropagation of Calluna vulgaris cv. ‘H.E. Beale’

Axillary shoot producing cultures were obtained from microcuttings and shoot tips of Calluna vulgaris cv. H.E. Beale. For cultures derived from microcuttings the highest multiplication rate of 38 shoots (5 mm or longer) was obtained on a reduced salt medium with the addition of 0.5 mgl^-1 2-isopentenyladenine (2iP) during an 8 week subculture. For shoot tip derived cultures 0.2 mgl^-1 6-benzyladenine (BA) was the best cytokinin and led to a multiplication rate of 26 for a 6 week subculture. The addition of 1 g/l casein hydrolysate to a multiplication medium enhanced shoot proliferation in presence of 0.5 mgl^-1 BA.Despite various auxin treatments shoots formed no roots in vitro but rooted readily if transferred to a peat substrate ex vitro. A high rooting percentage (80%) was also obtained with shoots taken from the end of a multiplication phase and rooted directly. An additional subculture on low auxin containing media before transfer to peat substrate is recommended because the shoot condition can be improved in this way. A high number of rooted plantlets was produced, so the methods described will allow mass propagation.     

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     

Plant regeneration of red raspberry (Rubus idaeus) cultivars ‘Joan J’ and ‘Polana’

In Vitro Cellular & Developmental Biology - Plant - Plants were regenerated from in vitro leaf and petiole tissues of Rubus spp. L. (red raspberry) ‘Joan J’ and ‘Polana’...     

Flavanols,as Plant Growth Inhibitors from Roots of Peach,Prunus persica Batsh. cv. ‘Hakuto ’

Three flavanols, which inhibit root growth in the rice seedling test, have been purified. One was (+)-afzelechin (1) and the other two, named Pia and PIb, were new biflavanols shown by the structures 2 and 3 on the basis of chemical and spectroscopic evidence. PIa inhibited the root growth of the peach seedlings. In addition, PIa was found in the soil in the area where the peach trees were growing. The flavanols, therefore, are suggested to be one of the chemical factors causing soil sickness often encountered in peach cultivation.     

Effects of CEPA and 1-MCP on Flower Bud Differentiation of Apple cv. ‘Nagafu No.2’ Grafted on Different Rootstocks

Journal of Plant Growth Regulation - The apple (Malus domestica Borkh.) has a relatively long sapling stage which prevents fruit breeding. The understanding of the flowering system is important to...