Plantlet formation from isolated protoplasts ofSolanum melongena L.

Summary Mesophyll protoplasts were isolated from axenic shoot cultures ofSolanum melongena by the one-step enzymatic method. Of the different media employed for the culture of protoplasts, a medium modified fromKao andMichayluk (1975) supported sustained mitotic cycles most effectively. Organogenesis from protoplast-derived callus was achieved on transfer toMurashige andSkoog'S (1962) medium supplemented with an appropriate auxin and a cytokinin.     

Callus induction and plantlet regeneration inCichorium intybus L.: II. Effect of different hormonal treatments

Summary Expiants ofCichorium intybus L. storage roots were grownin vitro on a modified Heller's medium lacking auxins and cytokinins, or supplemented with auxins (either 2,4-D or NAA) alone or with a cytokinin (kinetin) or auxin and kinetin combinations in different concentrations. The morphogenetic responses of root explants varied with the different hormonal treatments. The best response for callus growth was obtained in presence of 2,4-D. On the contrary, kinetin alone was very effective for shoot induction, increasing the formation of adventitious buds (up to 100% of the explants) in respect to control (hormone-free medium). NAA induced either shoot differentiation (in a medium frequency) or root formation. Expiants excised from root zones near to apex, which showed on hormone-free medium a very low regenerative capacity (lower than proximal zones of the root), responded to kinetin by increasing significantly the number of shoots from adventitious buds.Cytological analyses in developing primary calli showed, in all media, high incidence of amitotic phenomena confirmed by DNA cytophotometry in calli at different growth stages. The histological analysis demonstrated the formation of meristematic growth centers on the organogenesis inducing media and the subsequent development of these meristemoids as shoot (or root) apices in the callus mass.The results are discussed in comparison with previous observations of the authors inCichorium intybus (Caffaro et al. 1982) and in relation to the action of hormonal treatments on callus formation and organogenesis. The cytological and histological results are also discussed in relation to the hormonal composition of the medium.     

Plant regeneration from callus and explants of Sesbania spp.

Root, hypocotyl and cotyledon explants of Sesbania bispinosa, Sesbania cannabina, Sesbania formosa, and Sesbania sesban were cultured on Murashige and Skoog medium with benzyladenine (BA; 2.22, 4.44, 8.88 M) in combination with 2,4-dichlorophenoxyacetic acid (2,4-d; 2.26, 4.52, 9.05 M), indolebutyric acid (IBA; 0.25, 0.49, 4.92 M) or naphthaleneacetic acid (NAA; 2.69, 5.37, 10.74 M). Although all explant types developed some callus, callus occurred earliest and continued to grow fastest with hypocotyls. Media including 2.4-d or NAA gave the fastest growing callus. Callus was subcultured up to 10 times at 20-day intervals and retained a rapid growth rate. Shoots regenerated readily from both hypocotyls or cotyledons but not from roots. Shoot organogenesis was most frequent with IBA (0.25–4.92 M) in combination with BA (4.44–8.88 M) and did not occur with 2,4-d. With each species at least one medium induced shoot differentiation from more than 50 percent of the callus pieces. With one exception, media containing IBA that induced shoot organogenesis on explants also did so in callus, but media containing NAA, even when effective with explants, did not cause differentiation of callus. Shoots that differentiated were excised and cultured on MS medium without growth regulators or with IBA (2.46, 4.92, 9.84 M). Roots developed after 3–8 days on an appropriate rooting medium, often without IBA. Rooted plantlets were transplanted to pots in a greenhouse and developed into normal plants. Suitable media and protocols for initiating and subculturing callus and regenerating whole plants in vitro from callus and explants have thus been established for four species of Sesbania.     

Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (<Emphasis Type="Italic">Pinus virginiana</Emphasis> Mill.)

Plant tissue necrosis and subsequent cell death are usually observed during in vitro regeneration in conifers, especially in plant regeneration via somatic organogenesis in pine species. Cell death is correlated with the elevated levels of peroxides. In this investigation, the effects of antioxidants on in vitro regeneration of Virginia pine (Pinus virginiana Mill.) were evaluated. Antioxidants, polyvinylpolypyrrolidone (PVPP) and 1,4-dithio-dl-threitol (DTT), were found to improve callus formation, shoot differentiation and growth, and shoot rooting by inhibiting tissue necrosis during the initiation of cultures and subculture of shoots. These treatments enabled the recovery and regeneration plants at high frequency through somatic organogenesis. Compared to the control, the frequencies of callus formation, shoot growth, and shoot rooting increased 15, 26, and 19%, respectively, by addition of 5 g/l PVPP and 2 g/l DTT. Higher peroxidase activity of tissue cultures during subculture from callus proliferation medium to shoot differentiation medium and to rooting medium was observed. The addition of antioxidants reduces and inhibits browning by reducing the accumulation of peroxidase.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - DTT 1,4-Dithio-dl-threitol - IBA Indole butyric acid - NAA -Naphthaleneacetic acid - PVPP Polyvinylpolypyrrolidone     

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.     

Induction of somatic embryos in pea,Pisum sativum L.

Using low concentrations of picloram (0.06 mg/l), embryoids were formed on the surface of leaf-derived callus of pea, Pisum sativum L. (c.v. Dippes Gelbe Victoria) upon transfer to liquid medium. After some days in culture, embryoids spontaneously separated from the calli, and developed into torpedo-shaped embryos, which were transferred to solid medium. In a second series of experiments, embryos were also formed by mutant 489C and a genetic line of Pisum arvense, which additionally exhibited embryogenesis also from epicotyl-derived callus. Some of the embryos showed root formation, but no shoot morphogenesis occurred. In a limited number of cases, an additional root was formed in the apparent shoot apical region after 2–5 days.     

In vitro regeneration of Vigna unguiculata (L.) Walp. cv. Blackeye cowpea via shoot organogenesis

An in vitro regeneration system was developed in cowpea [Vigna unguiculata (L.) Walp.] Blackeye. Among several explants studied, shoot initiation response was observed from shoot apices of 3–5-day-old seedlings. The optimal medium for maximum shoot initiation comprised MS salts, B₅ vitamins, 8.88 μM N ⁶-benzylaminopurine, 1 gl^-1 casein hydrolysate, 342 μM L-glutamine, 3% sucrose, 0.3% phytagel, adjusted to pH 5.8. A shift in pH from 5.8 to 7.0 had no effect on shoot initiation and on number of shoots per explant. The highest shoot initiation frequency (77%) was obtained using this preferred medium, reaching a maximum of eight shoots per explant. For shoot elongation, 14 μM gibberellic acid was supplemented in the shoot initiation medium. Presence of indolebutyric acid in the rooting medium had no effect on root induction. The regenerated plants were fertile and developed normally.     

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.     

A novel approach to the establishment of dual cultures of pearl millet and Sclerospora graminicola

Dual cultures were successfully established using malformed florets of pearl millet infected with Sclerospora graminicola, the downy mildew pathogen. A higher proportion (86%) of calli from malformed florets formed dual cultures on Murashige and Skoog's (MS) medium with 2 mg 1^-1 of 2,4-dichlorophenoxy acetic acid (2,4-d), compared to shoot tips (25%). Fungal mycelium covered the entire surface of the callus within 30 days of placement of explants on the MS medium with 2 mg 1^-1 of 2,4-d. The infected calli also differentiated and produced plantlets when transferred to MS medium without 2,4-d.     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

Efficient plant regeneration from leaves of rapeseed (Brassica napus L.): the influence of AgNO3 and genotype

Factors influencing reliable shoot regeneration from leaf explants of rapeseed (Brassica napus L.) were examined. Addition of AgNO₃ to callus induction medium was significantly effective for shoot regeneration in all three genotypes initially tested. When 48 genotypes subsequently were surveyed, a large variation of shoot regenerability was observed, ranging from 100 to 0% in frequency of bud formation and from 7.5 to 0 in the number of buds per explant. A significant correlation (r=0.84) was observed between the frequency of bud formation and the number of buds per explant. The shoot regenerability from leaf explants was not related to that from cotyledonary explants (r=0.28). Histological observations showed that an organized structure developed from calluses produced at vascular bundle tissues after 7 days of culture on callus induction medium, and they developed shoot apical meristems one week after transfer onto shoot induction medium. Regenerated plantlets were obtained 2 months after the initiation of culture and they normally flowered and set seeds. No alterations of morphology or DNA contents were observed in regenerated plants and their S1 progenies.     

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l^-1), myoinositol (100 mg l^-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 M : 0.02 d^-1) and NAA (5.4 M : 0.06 d^-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 M) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 M)+NAA (0.54 M); Zeatin (45.62 M)+NAA (5.37 M) or BA (8.9 M) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 M)+NAA (1.08 M) or BA (13.32 M) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW^-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.     

Regeneration of salvia (<Emphasis Type="Italic">Salvia officinalis</Emphasis> L.) via induction of meristematic callus

Nodular meristematic callus was induced on the basal cut surface of apical shoot explants of salvia cultured on Murashige and Skoog (MS) medium supplemented with 4.5, 13.5, or 22.5 μM thidiazuron (TDZ). Cultures were incubated in the dark for 1 wk and then transferred to light conditions for 4 wk. A higher percentage of explants developing callus was observed on medium containing either 4.5 or 13.5 μM TDZ, although explants on 4.5 μM developed larger calluses. The callus was maintained on medium containing 4.5 μM TDZ and 0.45 mM ascorbic acid. Shoot differentiation, after each of three successive maintenance passages, was induced from callus grown on medium containing either 4.4 or 8.8 μM benzyladenine (BA). A greater number of shoots were harvested from callus differentiated on BA (4.4 or 8.8 μM) medium with 0.45 mM ascorbic acid added. Shoots developed roots on MS medium supplemented with 4.9 μM of indole-3-butyric acid. The addition of ascorbic acid to the shoot differentiation medium enhanced rooting, number of roots per shoot, and survival rate. Approximately 75% in vitro plantlets were acclimatized to ex vitro conditions. Histological investigations confirmed both adventitious meristem initiation during the callus induction phase, and subsequent organogenic shoot development on the differentiation medium. The novel protocol for the meristematic callus induction and plant regeneration in this study may be useful for biotechnological applications for salvia improvement via genetic transformation or mutagenesis and in vitro propagation approaches.     

Regeneration of plants from seed-derived callus of Hybanthus enneaspermus L. Muell., a rare ethnobotanical herb

A procedure was developed for plant regeneration of Hybanthus enneaspermus, a rare ethnobotanical herb from the Deccan peninsula in India, through seed-derived callus. Seeds demonstrated a high induction frequency (69.4±2.8%) and a high yield (364.4±2.5 mg) of light-yellow friable callus on Murashige and Skoog's (MS) medium containing 2.6 μm NAA and 2.2 μm BA within 4 weeks of incubation. After 1 year of subculture, yellow friable and light-green compact calli types were established from initial light-yellow friable callus. Shoot differentiation was achieved from light-green compact callus, but not from yellow friable callus. Shoot differentiation resulted when light-green compact callus was transferred to MS medium supplemented with 8.8 μm BA and 2.6 μm NAA; the highest percentage of calli forming shoots (66.6±4.8%) and the highest number of shoots (8.9±0.3) were achieved in this medium. Differentiated shoot buds elongated to 4–5 cm within 4 weeks. The addition of casein hydrolysate (500 mg/l) and more potassium phosphate (1.86 mm) to the culture medium enhanced shoot differentiation. Rooting was achieved on the shoots using half-strength MS medium containing 4.8 μm IBA. About 70% of the plants were established in pots containing pure garden soil after 2 weeks of hardening. The regenerated plants were morphologically uniform and exhibited normal seed set. Received: 23 July 1998 / Revision received: 18 November 1998 / Accepted: 26 November 1998     

Isolation and culture of protoplasts ofCapsicum annuum L. and their regeneration into plants floweringin vitro

Summary Axenic shoot cultures ofCapsicum annuum cv.California Wonder were used as the source for isolation of protoplasts from mesophyll cells. Protoplasts underwent sustained mitotic activity and proliferated to form callus masses on NT or DPD medium enriched with 2,4-D, NAA and BAP each at 1 mg/l level. The callus could be differentiated into whole plants on the differentiation media and plants floweredin vitro under long day conditions.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of the heavy metal tolerant and hyperaccumulator <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> (Brassicaceae)

Plants were regenerated from root explants of Arabidopsis halleri (L.) O’Kane and Al-Shehbaz via a three-step procedure callus induction, induction of somatic embryos and shoot development. Callus was induced from root segments, leaflets and petiole segments after incubation for 2 weeks in Murashige and Skoog medium (MS) supplemented with 0.5 mg/l⁻¹ (2.26 μM) 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.05 mg/l⁻¹ (0.23 μM) kinetin. Only calli developed from root segments continued to grow when transferred to a regeneration medium containing 2.0 mg/l⁻¹ (9.8 μM) 6-γ-γ-(dimethylallylamino)-purine (2ip) and 0.05 mg/l⁻¹ (2.68 μM) α-naphthalenacetic acid (NAA) and eventually 40 of them developed embryogenic structures. On the same medium 38 of these calli regenerated shoots. Rooting was achieved for 50 of the shoots subcultured in MS medium without hormones. The regeneration ability of callus derived from root cuttings, observed in this study, makes this technique useful for genetic transformation experiments and in vitro culture studies.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and plant establishment of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. F.) Merrill: Petiole callus culture

Summary A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented with 10μM2,4-dichlorophenoxyacetic acid +2,5μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5μM TDZ. Individual elongated shoots were rooted best on halfstrength MS medium containing 0.5μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in a greenhouse with 100% survival. Four months after transfer to pots, the performance of in vitro-propagated plants of T. indica was evaluated on the basis of selected physiological parameters and compared with ex vitro plants of the same age.     

Callus production from willow (Salix viminalis L.) protoplasts

Protoplasts were isolated from cell suspensions of Salix viminalis (basket willow) clone 78-0-90 and S. schwerinii clone 77-0-77, using cellulysin and macerase in modified Woody Plant medium. For clone 78-0-90, 6.3 · 10⁶ ± 1.9 · 10⁶ protoplasts were obtained per gram fresh weight. Cell divisions started two days after protoplast isolation and gave rise to callus which has been maintained in culture for up to four years. Protoplast yield from the clone 77-0-77 was lower (less than 10⁶ protoplasts per gram cells), cell division was infrequent and no callus was obtained. Protoplasts were also isolated from the leaves of willow shoot cultures using cellulysin and pectolyase, but these did not show cell divisions.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS medium Murashige & Skoog (1962) medium - WP medium Woody Plant medium (Lloyd & McCown 1981)     

Continuous callus production and regeneration of garlic (Allium sativum L.) using root segments from shoot tip-derived plantlets

Root segments from shoot tip-derived plantlets of the garlic (Allium sativum L.) clones `DDR7099', `PI383819', and `Piacenza' were utilized as an explant source for continuous, friable callus production. The best callus production occurred on root segments initially cultured on medium with 4,5 μm 2,4-dichlorophenoxyacetic acid (2,4-D) for 8 weeks, then subcultured to medium with 4.7 μm 4-amino-3,5,6-trichloropicolinic acid (picloram) +0.49 μm 6-(γ-γ-dimethylallylamino)purine (2iP) for 8 weeks. Embryogenic, friable callus was transferred to liquid medium for 1 month and then transferred to solid regeneration medium for 14 weeks. The best shoot and root regeneration (85.3% and 35.8%, respectively) occurred on 4-month-old calli from the clone `DDR7099'. In all clones, regeneration rate decreased as callus age increased. Received: 14 October 1997 / Revision received: 26 December 1997 / Accepted: 12 January 1998