In vitro differentiation of multiple shoot clumps from intact seedlings of switchgrass

Summary An efficient and reproduciblein vitro culture system has been developed for regeneration of multiple shoot clumps from intact seedlings of both lowland and upland cultivars of switchgrass (Panicum virgatum L.). The multiple shoots were induced on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 1-phenyl-3-(1,2,3-thiadiazol-5YL)-urea (thidiazuron or TDZ). Maximum response was obtained with 4.5 μM 2,4-D and 18.2 μM TDZ. These shoots proliferated and rooted efficiently on MS medium without growth regulators. The developmental pattern of the multiple shoots indicated their origin from the enlarged shoot apex via proliferation of axillary buds and subsequent reprogramming of shoot meristems followed by secondary differentiation of adventitious shoots The simplicity of the protocol and direct production of multiple shoots make this a potential system that is highly attractive and amenable for microprojectile-mediated gene transfer.     

Thidiazuron stimulates shoot regeneration of sugarcane embryogenic callus

Summary Efficient shoot regeneration of sugarcane (Saccharum spp. hybrid cv. CP84-1198) from embryogenic callus cultures has been obtained using thidiazuron (TDZ). Callus was placed on modified Murashige and Skoog (MS) medium containing 2.3 μM 2,4-dichlorophenoxyacetic acid (2,4-D), or 9.3 μM kinetin and 22.3 μM naphthaleneacetic acid (NAA) and compared with the same MS medium supplemented with 0.5, 1.0, 2.5, 5.0 or 10.0 μMTDZ, A11 TDZ treatments resulted in faster shoot regeneration than the kinetin/NAA treatment, and more shoot production than either the 2,4-D or kinetin/NAA treatments. Maximum response, as determined by total number of shoots (26 per explant) and number of shoots greater than 1 cm (4 per explant) 4 wk after initiation, was obtained with 1.0 μM TDZ. The shoots rooted efficiently on MS medium supplemented with 19.7 μM indole-3-butyric acid (IBA). These results indicate that TDZ effectively stimulates sugarcane plant regeneration from embryogenic callus, and may be suitable to use in genetic transformation studies to enhance regeneration of transgenic plants.     

Adventitious regeneration of Juglans nigra L. (eastern black walnut)

Somatic embryos and adventitious shoots were initiated from immature cotyledons 10–14 weeks after anthesis. Maximum embryogenesis occurred 12 weeks after anthesis and maximum shoot organogenesis occurred 14 weeks after anthesis. The best treatment for induction of somatic embryos and adventitious shoots from immature cotyledon explants was on agar-solidified WPM supplemented with 0.1 M 2,4-D and 5.0 M TDZ and incubated in light for the first four weeks. Rooting of adventitious shoots was best if they were quickdipped in 2.5 mM IBA and 1.25 mM NAA in 1% dimethyl formamide and 3.9% ethanol (120 Wood's Rooting Compound: water, by volume). Plantlets from rooted adventitious shoots were acclimatized to the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - DKW Driver and Kuniyuki (1984) walnut medium - IBA indolebutyric acid - LP Long and Preece medium described herein - NAA naphthaleneacetic acid - PPF photosynthetic photon flux - TDZ thidiazuron - WPM Woody Plant Medium of Lloyd and McCown (1980)     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

Transformation of sweetgum via microprojectile bombardment of nodule cultures

Summary A sweetgum (Liquidambar styraciflua) nodule culture system was developed and integrated with genetic transformation by microprojectile bombardment. Nodule cultures were established from seedling hypocotyls and proliferated in liquid medium containing 0.1 mg (0.45 μM) thidiazuron (TDZ) per 1 and 0.01 mg (0.045 μM) 2,4-dichlorophenoxyacetic acid (2,4-D) per 1. Shoots differentiated from the nodules in liquid media containing (per 1) 1 mg (4.4 μM) benzyladenine (BA), 0.5 mg (2.2 μM) BA, and 0.01 mg (0.054 μM) naphthaleneacetic acid (NAA), or 0.5 mg BA, 0.01 mg NAA, and 0.05 mg (0.23 μM) TDZ under the light. Differentiating shoots required 4 wk of dark treatment for further development on semisolid medium containing 1 mg BA per 1. Elongated shoots were harvested and the basal ends were soaked in a solution containing 10 mg (49.2 μM) indole-3-butyric acid (IBA) per 1 before being planted in potting mix for ex vitro rooting. Roots formed and leaves expanded in 2 wk. Sweetgum nodules were stably transformed by microprojectile bombardment with a 7.4-kb plasmid, pTRA 140, harboring CaMV 35S-HPH and CaMV 35S-GUS. Evidence that nodules growing in the presence of hygromycin B were stably transformed was provided by polymerase chain reaction analysis and β-glucuronidase activity. Sweetgum shoots differentiated in liquid medium in the presence of hygromycin B. Shoots transferred to solid medium lacking hygromycin B elongated and displayed β-glucuronidase activity in their expanding leaves and stems. Southern analysis confirmed the presence of the GUS gene in nodules and shoots. Transgenic shoots initiated roots and showed leaf expansion 2 wk after being planted in potting mix.     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

Micropropagation of Gymea Lily (<Emphasis Type="Italic">Doryanthes excelsa</Emphasis> Corrêa) from New South Wales,Australia

The physiological effects of three auxins [indole-3-butyric acid (IBA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d)] and two cytokinins [thidiazuron (TDZ) and N⁶-benzylaminopurine (NAA)] on in vitro morphogenesis of Doryanthes excelsa were measured. Longitudinal bud sections derived from immature inflorescences were used as a source of explants. Callus regeneration was observed at the highest frequencies (46.2%) when grown on media containing 50 μmol L^-1 NAA and 0.5 μmol L⁻¹ TDZ. Adventitious shoot organogenesis was observed at the highest frequency (56.8%) when grown on media containing 0.5 μmol L⁻¹ NAA and 50 μmol L⁻¹ TDZ. Regenerated shoots were rooted ex vitro after 6 weeks when dipped in a solution of 50 μmol L⁻¹ NAA or no plant growth regulators were applied.     

Improvement of somatic embryogenesis in highland-papaya cell suspensions

Axillary buds (2 mm) from 3-year-old Carica pubescens Lenné et Koch (highland papaya) fruit-bearing plants grown in the greenhouse were cultivated in NN-medium supplemented with different growth regulators naphthaleneacetic acid and indoleacetic acid in combination with Zeatin, benzyladenine, Kinetin and thidiazuron. Several responses were observed within 2–3 months; namely, sprouting of the preformed axillary buds, bud branching into multiple shoots, callus formation at the basal end of the explant and somatic embryogenesis in the preformed callus. Somatic embryogenesis was frequent in most of the tested growth regulator combinations, with the exception of thidiazuron which showed no effect. A much higher yield of somatic embryos could be obtained in suspensions. Somatic embryogenesis was enhanced by the occurence of adventive embryogenesis on single embryos as globular embryo clusters. This was observed in cell suspensions initially grown in a WPM-medium with 2,4-dichlorophenoxyacetic acid, or in combination with benzyladenine or zeatin, for 6 days, then maintained in a growth regulator-free medium under continuous agitation (50 RPM) on an orbital shaker for 3 months. Single cells grown in the absence of 2,4-dichlorophenoxyacetic acid did not initiate embryogenesis and de-differentiated into callus. Plantlets were recovered after transfer of mature embryos from cell suspensions into Magenta flasks. In a second subculture, adventitious embryogenesis occurred spontaneously in clusters at the globular embryo stage under the same growth conditions, yielding a high number of embryos. The culture conditions described above allowed initiation of a large number of somatic embryos directly from cell suspensions through adventive somatic embryogenesis and indirectly from callus on axillary buds.Abbreviations 2,4-d dichlorophenoxyacetic acid - CH casein enzymatic hydrolysate - BA benzyladenine - FAA formalin:acetic acid:alcohol - Glu l-glutamine - IAA indoleacetic acid - NAA naphthaleneacetic acid - NN Nitsch and Nitsch-medium (1969) - TDZ thidiazuron - SD standard deviation     

Adventitious shoot regeneration from leaf explants of almond (Prunus dulcis mill.)

Summary A method has been developed to facilitate shoot formation from leaf explants of almond. Leaves were dissected from micropropagated shoot cultures of the commercial cultivars Nonpareil and Ne Plus Ultra, and sections incubated on Almehdi and Parfitt's (1986) basal medium (AP) with varied plant growth-regulator conditions. Three auxins, 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA), in combination with two cytokinins, benzylaminopurine (BA) and thidiazuron (TDZ), were tested at various concentrations along with casein hydrolysate (CH) to determine, the conditions most conducive to adventitious shoot regeneration. Response to the tested plant growth-regulator conditions varied with genotype. Of the three auxins tested, NAA and IBA induced adventitious shoots from Ne Plus Ultra explants, but only IBA was effective for Nonpareil. For the cytokinins, shoot development from Ne Plus Ultra occurred in the presence of either BA or TDZ, whereas for Nonpareil only TDZ was effective unless CH was incorporated in the basal medium. The inclusion of CH (0.1% w/v) improved callus morphology, and increased regeneration frequencies for both cultivars. Maximum regeneration frequencies for Ne Plus Ultra (44.4%) and Nonpareil (5.5%) were achieved on AP basal salts supplemented with CH, IBA (9.8 μM), and TDZ at 22.7 and 6.8 μM, respectively.     

Plantlets from somatic callus tissue of the East Indian Rosewood (Dalbergia latifolia Roxb.)

Callus-mediated shoot bud formation was demonstrated in Dalbergia latifolia Roxb. (East Indian Rosewood). Cultures were raised from shoot explants of six year-old plants on Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA) and benzyladenine (BA). A sequential treatment of callus with increasing BA levels and decreasing NAA ensured shoot bud induction. Rooting of shoots was achieved by a three-step culture procedure involving 1) White's(W) liquid medium containing indoleacetic acid (IAA), naphthaleneacetic acid and indolebutyric acid (IBA), 2) half-strength MS agar-solidified medium with charcoal (0.25%) and 3) half-strength MS liquid medium.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - MS Murashige and Skoog - NAA a-naphthaleneacetic acid - PVP Polyvinylpyrrolidone - W White's medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

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.     

A highly efficient plant regeneration system through multiple shoot differentiation from commercial cultivars of barley (Hordeum vulgare L.) using meristematic shoot segments excised from germinated mature embryos

A fast and highly efficient short-term in vitro regeneration system was developed for barley (Hordeum vulgare L.) based on readily available explants. Clumps of multiple shoots and buds suitable for transformation were obtained 9–10 weeks after culture initiation from model and current commercial cultivars. Meristematic shoot segments (MSSs) excised from mature embryo-derived seedlings and subsequently cultured on MS-based medium containing 2 mg/l Picloram and 3 mg/l thidiazuron (TDZ) differentiated up to ten multiple shoots after 3–4 weeks with no or very little callus formation. Sectors of the already multiplied shoot clumps were further multiplied on proliferation-maintenance medium containing 2 mg/l Picloram and 2.5 mg/l TDZ. Biweekly subcultures resulted in a continuous process of multiplication of these highly differentiating green sectors without any loss of morphogenic potential. The differentiated small shoots and shoot buds gave rise to normal shoots on medium with 0.1 mg/l Picloram and 1 mg/l TDZ. After rooting on basal medium with 0.5 mg/l or 1 mg/l IBA the plants were transferred to soil and showed normal growth and fertility compared to the seed-grown plants. All of the genotypes tested formed multiple shoots. The percentage of relative MSS multiplication was 63–83%, and the average number of multiplied shoots per MSS ranged from 16 to 34 among the genotypes after 9–11 weeks.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - Dicamba 3,6-Dichloro-2-methoxybenzoic acid - IBA Indole-3-butyric acid - MSS Meristematic shoot segment - NAA -Naphthaleneacetic acid - Picloram 4-Amino-3,5,6-trichloropicolinic acid - TDZ Thidiazuron     

Shoot Organogenesis from Immature Zygotic Embryo Cultures of Ginkgo biloba

Immature zygotic embryos were cultured on Murashige and Skoog's medium (MS) supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA), benzyladenine (BA) and zeatin or with various concentrations of 2,4-D alone. The maximum number (8 per embryo) of adventitious buds formed from cotyledons of heart stage embryos cultured on MS medium with 1 mg dm⁻³ BA and 0.01 mg dm⁻³ NAA. The adventitious buds originated from procambial strands of immature embryo cotyledons and then developed into adventitious bud primordia within 20 d. Adventitious buds transferred to hormone free MS medium grew into shoots, but did not produce plantlets because the shoots failed to root. This revised version was published online in July 2006 with corrections to the Cover Date.     

In vitro regeneration of evergreen azalea from leaves

Rhododendron simsii Hellmut Vogel was regenerated using different types of explants, auxins and cytokinins. After a callus induction phase, with 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid, adventitious shoot regeneration was obtained on a medium supplemented with thidiazuron or zeatin. With thidiazuron shoots were small and a subsequent elongation step was required before rooting. An elongation step was not required when zeatin was used. The duration of the callus induction phase was negatively correlated with the regeneration capacity.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - 2iP 6-(--dimethylallylamino)purine - NOA ß-naphthoxyacetic acid - BA 6-benzyladenine - IBA 1-H-indole-3-butyric acid - IAA 1-H-indole-3-acetic acid - TDZ thidiazuron - WPM woody plant medium     

Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.)

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 M thidiazuron but not on explants on media with benzyladenine (BA) or isopentenyladenine. Not all seed sources were equally capable of shoot organogenesis and embryogenesis. Atypical of adventitious regeneration of other woody plants, mature seed explants of white ash were more organogenic with shoots that elongated better than explants from immature seeds. Somatic embryogenesis was observed in cultures where mature seeds were first cultured for 4 weeks on a medium containing 10 M adenine 2,4-dichlorophenoxyacetic acid in combination with 0.1 and 1.0 M thidiazuron, followed by transfer to a medium containing 0.05 M 6-benzyladenine and 0.5 M naphthaleneacetic acid. Adventitious shoots and epicotyls from both seedlings and germinated somatic embryos were rooted under intermittent mist and acclimatized to the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - TDZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-ylurea - WPM woody plant medium     

Direct somatic embryogenesis and shoot regeneration from leaves and internodes of Pluchea lanceolata (DC.) C.B. Clarke

Pluchea lanceolata (DC.) C.B. Clarke is a threatened native medicinal plant. Increasing the propagation of this plant will preserve the wild population and provide material for medicinal use. In vitro and field-collected shoots and leaves were tested for response to 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), for initiation of direct shoot regeneration (DSR), or direct somatic embryogenesis (DSE). Leaves and internodes collected from field-grown plants produced only callus, while in vitro–raised shoots exhibited DSR and DSE on Murashige and Skoog (MS) medium with 2,4-D and TDZ. Direct shoot regeneration occurred on medium with TDZ from internode and leaf segments obtained from in vitro–developed shoots. In vitro–grown shoots were rooted on half-strength MS medium with 2 mg L⁻¹ indole-3-butyric acid and acclimatized. Survival in natural conditions was 62.5% for DSE and 79% for DSR plantlets.

     

The effect of in vitro pretreatments on subsequent shoot organogenesis from excised Rubus and Malus leaves

Shoot regeneration from Rubus leaves was obtained on a medium containing MS salts, vitamins and sugars, Staba vitamins, casein hydrolysate (100 mg l^–1) and 10 M thidiazuron. Shoot regeneration from Malus leaves was obtained on N⁶ rice anther medium with 5 M thidiazuron. In vitro pretreatment of source shoots with either colchicine or thidiazuron enhanced the organogenic potential of detached leaves of two Rubus hybrids. The response to colchicine was quadratic and occurred at non-mutagenic concentrations (75–250 M). The response to thidiazuron was exponential between 0 and 5 M. When applied as a pretreatment, the effectiveness of several different cytokinins (benzyladenine, thidiazuron, zeatin) at enhancing Malus and Rubus organogenesis was related to the shoot proliferation activity of the cytokinin and to treatment-induced variation in leaf and petiole size.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - MS Murashige & Skoog basal medium devoid of plant growth regulators - OI organogenesis-initiating subculture - PTI colchicine pretreatment subculture - PTII cytokinin pretreatment subculture - NAA naphthaleneacetic acid - TDZ thidiazuron - zeatin trans-zeatin     

Transformation of <Emphasis Type="Italic">Liquidambar formosana</Emphasis> L. via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> using a mannose selection system and recovery of salt tolerant lines

Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l⁻¹ induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l⁻¹ α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant (14.6) was observed on MS medium with 3 mg l⁻¹ TDZ, 0.5 mg l⁻¹ NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium with 3 mg l⁻¹ BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with 4 mg l⁻¹ indole-3-butyric acid (IBA) and 1.5 mg l⁻¹ activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot regeneration of the medicinal plant meadowsweet (<Emphasis Type="Italic">Filipendula ulmaria</Emphasis> (L.) Maxim)

Filipendula ulmaria (L.) Maxim (meadowsweet) is a medicinal plant that is claimed to have several biological activities, including anti-tumor, anti-carcinogenic, anti-oxidant, anti-coagulant, anti-ulcerogenic, anti-microbial, anti-arthritic, and immunomodulatory properties. This report describes, for the first time, an efficient plant regeneration system for F. ulmaria via adventitious shoot development from leaf, petiole, and root explants cultured on Murashige and Skoog’s minimal organics medium containing different concentrations of thidiazuron (TDZ), benzyladenine, and kinetin either alone or in combination with different auxins. Relatively extensive/prolific shoot regeneration was observed in all three explant types with TDZ in combination with indole-3-acetic acid (IAA). Gibberellic acid (GA₃), TDZ, and IAA combinations were also tested. The best shoot proliferation was observed among root explants cultured on media supplemented with 0.45 μM TDZ + 2.85 μM IAA + 1.44 μM GA₃. Regenerated shoots were transferred to rooting media containing different concentrations of either IAA, indole-3-butyric acid (IBA), naphthalene acetic acid, or 2,4-dichlorophenoxyacetic acid. Most shoots developed roots on medium with 2.46 μM IBA. Rooted explants were transferred to vermiculite in Magenta containers for a 2-wk acclimatization period and then finally to plastic pots containing potting soil. The plantlets in soil were kept in growth chambers for 2 wk before transferring to greenhouse conditions.     

Plant regeneration of grapevine (Vitis sp.) protoplasts isolated from embryogenic tissue

Summary Protoplasts with high embryogenic competence could be isolated from leaf-disk-derived embryos and embryoids of Vitis sp. cv. Seyval blanc. After a 4-week induction treatment in NN-69 medium supplemented with 4.0mg/l naphthoxyacetic acid (NOA) and 0.9mg/l thidiazuron (TDZ) and subsequent subcultivation in hormone-free medium, 38.5% of the developed microcalluses showed somatic embryogenesis. In contrast, only few formed somatic embryos after induction in CPW-13 medium with either 1.0mg/l 2,4-dichlorophenoxyacetic acid and 0.5mg/l benzylaminopurine treatment (13.8%) or NOA/TDZ treatment (1.4%). Up to 30% of these embryos germinated and about half of them regenerated into typical in vitro grapevines when transferred onto LS-medium in culture tubes.Abbreviations BAP benzylaminopurine - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-[N-morpholino]-ethanesulfonic acid - NOA naphthoxyacetic acid - PCR polymerase chain reaction - PVP polyvinylpyrrolidone - TDZ thidiazuron