Callus induction and culture from explants of Erysimum scoparium in a growth regulator-free medium

Calli from cotyledon, hypocotyl and radicle explants of Erysimum scoparium (Brassicaceae) were induced and cultured using a MS medium without growth regulators. Calli obtained from cotyledon or hypocotyl explants showed a higher growth rate while radicle-derived calli exhibited poor growth. Cotyledon-derived calli were compact and green-colored while hypocotyl- and radicle-derived calli were friable and with creamy-green pigmentation. Histological analysis was carried out during culture and the development of abundant vascular elements and meristematic nodules were found. Subculture on a growth regulator-free medium during 10 months did not ecrease callus growth.     

In vitro selection at cellular level for red rot resistance in sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> sp.)

In the present study, in vitro selection technique using pathogen culture filtrate of Colletotrichum falcatum Went was employed with the aim to identify associations (if any), between selection at the cellular and plant level for red rot resistance in sugarcane (Saccharum sp.). Five to eight months old sugarcane calli of genotypes CoJ 88 and CoJ 64 were screened in vitro against pathogen culture filtrate for two selection cycles. Effect of pathogen culture filtrate on callus survival and/or proliferation was observed to be directly related to its concentration in the selection media. Calli survived and exhibited further proliferation at 5, 10 and 15% v/v pathogen culture filtrate concentrations whereas, at higher concentrations (20 and 25% v/v) proliferation was completely inhibited. Shoot regeneration percent was higher in calli selected on 5% pathogen culture filtrate concentration than those selected on 10 and 15% concentrations. In vivo screening of field transferred somaclones against two pathtypes (Cf 03 and Cf 08) showed considerable variation for red rot resistance. Somaclones regenerated from resistant and/or tolerant calli exhibited better resistance than the parental genotypes. The results indicated that in vitro selection for red rot resistance was effective and expressed when somaclones were screened in the field. This indicated a positive association between in vitro and in vivo methods of selection for disease resistance in sugarcane.     

Callus induction and somatic embryogenesis of Phalaenopsis

Callus induction and plant regeneration through somatic embryogenesis in Phalaenopsis Richard Shaffer `Santa Cruz' were examined. Protocorm-like body (PLB) segments formed calli in Vacin and Went medium with sucrose. The optimal concentration of sucrose was 40 g ⋅ l^–1. Medium containing 200 ml ⋅ l^–1 coconut water together with 40 g ⋅ l^–1 sucrose was effective for callus induction. Gellan gum was suitable than agar as a gelling agent for callus induction. The calli easily formed PLBs after being transferred to a medium without sucrose. Histological observation suggested that the PLBs were somatic embryos. No variation was observed in the flowering plants regenerated through somatic embryogenesis. Received: 11 June 1997 / Revision received: 6 October 1997 / Accepted: 18 October 1997     

High frequency plant regeneration of garlic (Allium sativum L.) calli immobilized in calcium alginate gel

Calli obtained from a shoot-tip of garlic,Allium sativum L., were encapsulated using a calcium alginate gel. Some of the encapsulated calli were cultured on a 1/2 MS medium supplemented with 3% sucrose, 10⁻⁵ M kinetin, and 5×10⁻⁶ M NAA, whereas the remainder was stored for 40 days at 4°C. All the naked calli regenerated on the solid medium, while 95% of the encapsulated calli regenerated, and 88% of the encapsulated calli regenerated after 40 days of storage at 4°C. The capsule matrix delayed the germination time of the encapsulated calli, yet activated the shoot formation of the artificial garlic seeds. The shoot length of the encapsulated garlic calli was much longer than that of the naked garlic calli. The encapsulated garlic calli were dried in a laminar airflow cabinet and the conversion frequency of the dried artificial garlic seeds on a 1/2 MS medium remained at 93% with a water loss of less than 50%.     

Somatic embryogenesis in Ranunculus asiaticus L. hybr. thalamus cultivated in vitro

Calli derived from in vitro cultivated thalamus of Ranunculus asiaticus L. were initiated and maintained for 75 days on Murashige & Skoog's medium containing five concentrations of 2,4-d (0.1, 0.2, 0.4, 0.8, 1.6 mg l^-1). Embryoid differentiation occurred on calli initiated on 1.6 mg l^-1 2,4-d 75 days after subculture onto hormone-free medium. Calli which were initiated and maintained for 75 days on lower 2,4-d concentrations, then transferred to medium without hormones for 75 days, showed the first embryoids one month after further subculture on medium containing 0.05 mg l^-1 2,4-d. All the somatic embryos developed into plants, and 96% survived transplantation to in vivo growth conditions.     

Improvement of regeneration ability in Phleum pratense L. in vitro culture by dicamba

Calli were induced from mature caryopses of timothy grass (Phleum pratense L.) on MS medium (Murashige and Skoog 1962) supplemented with 500 mg·dm⁻³ casein hydrolysate and 5 mg·dm⁻³ 2,4-D (2,4-dicholorophenoxyacetic acid) or 2 mg·dm⁻³ dicamba (3,6-dichloro-o-anisic acid). Twelve-week-old calli were passaged on media with reduced levels of auxins (2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba). Tissues induced on medium with 2,4-D were transferred on medium with 2,4-D and on medium with dicamba; parallely calli initiated on medium with dicamba were passaged on medium with 2,4-D or dicamba. Calli from various media sequences were used to establish cell suspension cultures in media containing 2 mg·dm⁻³ 2,4-D or 1 mg·dm⁻³ dicamba. An assessment of regeneration ability of calli was made on MS medium containing 0.2 mg·dm⁻³ kinetin. Callus tissue induced and/or subcultured on any of the media with 2,4-D did not regenerate plants while dicamba added to the media was the effective stimulator of regenerability. In the presence of 2,4-D calli and suspensions produced a jelly-like extracellular matrix. In cell suspension this phenomenon was observed 4–5 days after each passage. The measurements of electric potential of calli, growing on MS medium with kinetin were performed. Non-regenerating callus areas had an electric potential close to 0 mV while parts of tissue with meristematic centres were characterized by lower values of electric potential.     

Callus and suspension cultures for biomass production ofCynara cardunculus (Compositae)

Summary Friable calli, obtained from hypocotyl and leaf segments ofCynara cardunculus, were used for the production of cell suspension cultures in liquid Gamborg B₅ medium supplemented as for callus obtention. The low inoculum cell suspension cultures (6.9×10⁴ cells.ml^–1) presented a td=136.8 hours while those obtained with a high inoculum (15.6×10⁴ cells.ml^–1) reached a td=33.6 hours.     

Somatic embryogenesis from leaf protoplasts of Rauvolfia vomitoria shoot cultures

Rauvolfia vomitoria mesophyll protoplasts have been isolated from axenic shoot cultures and cultured (10⁵-10⁶ protoplasts per ml) in Murashige and Tucker liquid medium containing growth regulators. Within 6–8 weeks, a mixed population of calli and proembryos were obtained and transferred on solid media. Calli produced shoots; however, rooting did not occur. Somatic embryos achieved different patterns of development. In particular, whole plantlets have been obtained either directly through germination of primary embryos or via embryogenic calli.Abbreviations B5 Gamborg et al. (1968) medium - BA N⁶ (benzyl) adenine - 2,4-D 2,4 dichlorophenoxyacetic acid - MT Murashige and Tucker (1969) medium - NAA naphthalene acetic acid - Z zeatin - K kinetin     

Changes in Sugars, Sucrose Synthase Activity and Proteins in Salinity Tolerant Callus and Cell Suspension Cultures of Brassica oleracea L.

Salt tolerant callus and cell suspension cultures of Brassica oleracea L. var. botrytis were obtained by the selection of cells from cultures growing in medium supplemented with 85, 170, and 255 mM NaCl. Salt adapted calli and cell suspensions differed in their RNA and protein concentrations. These concentrations tend to diminish in calli and increase in cell suspensions, both at one or three weeks periods of growth in NaCl. Contents of sucrose and reducing sugars, however, accumulate similarly both in calli and cell suspensions after NaCl treatments. The activity of sucrose synthase was higher in salt adapted cells than in controls. Calli exposed to 255 mM NaCl for six months synthesized a 27 kDa polypeptide, while a 13 kDa polypeptide present in control conditions was absent under salinity. Several high molecular mass polypeptides (> 200 kDa) were visualized in control calli and at moderate salt concentrations, when conditions of the gel were modified.     

High Frequency Plant Regeneration from Callus Culture of Pleione formosana Hayata

High frequency plant regeneration was induced from protocorm-derived callus cultured on half-strength of Murashige—Skoog medium with 2,4-dichlorophenoxyacetic acid (2,4-D, 0–5 mg l⁻¹) and 1-phenyl-3-(1,2,3-thiadiazol-5-yl, 0–1 mg l⁻¹) urea (TDZ) in the dark. Twelve totipotent callus lines were selected within 76 callus lines regenerated on half-strength of Murashige—Skoog (MS) medium with 0.5 mg l⁻¹ TDZ. The proliferation rate was 4–5-fold in fresh weight after 30 days of culture on half-strength MS medium containing 5 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ TDZ in the dark. The maximum number of shoot buds generated by 0.01 g callus explant was 134 after 4 months of culture. These calli were regenerated to plantlets via protocorm-like bodies (PLBs) after 75–150 days of culture. The shoots, with two true leaves, were transferred to hormone-free medium, rooting and eventually formed plantlets. Totipotent callus lines of Pleione formosana Hayata have been successfully established in this study.     

Somatic embryogenesis and organogenesis in callus cultures of a tree legume — Albizia richardiana King

Hypocotyl explants of 1 and 10 mm lengths were excised from 12-day-old in vitro-grown seedlings of Albizia richardiana. The larger pieces, after 40 days of culture, developed shoots along with green calli on B₅ + BAP (10^–7–10^–5M), while the smaller segments produced only green calli on B₅+BAP (10^–7–10^–4M) medium. Some of the green calli turned morphogenic and started producing somatic embryos with the 2nd sub-culture and shoots from 7th sub-culture onwards. Calli retained the morphogenic potential even after repeated sub-culturing for over two years. The number of embryos in an embryogenic culture varied from 2 to 20 per callus mass of 5–6.5 cm³. Sucrose at the 2% level in MS medium was optimal for embryogenesis while 4% was optimal for shoot bud differentiation. Higher levels of sucrose (6–10%) caused browning of green calli and also inhibited differentiation into embryos and shoot buds. By selective sub-culturing of 0.1 cm³ pieces of embryogenic calli on MS+10^–5M BAP, 46% of the cultures produced somatic embryos. The latter germinated into plantlets on Knop's medium.Abbreviations BAP 6-benzylaminopurine - B₅ Gamborg et al., 1968 medium - IAA Indole-3-acetic acid - MS Murashige and Skoog's (1962) medium     

Morphogenesis in Tissue Cultures of Different Organs of Nigella sativa

Calli were isolated from root, stem and leaf segments of Nigella sativa (Fam. Ranunculaceae) on White's medium containing napthaleneacetic acid and coconut milk. From all the three types of calli, roots were differentiated. Shoot development occurred in stem and leaf calli only after the omission of first coconut milk and then of auxin from the basal medium and also in IAA (2.0 mg/ml) and coconut milk (15 % v/v) in the medium. Frequency of organ formation and the maintenance of this capacity depend upon the nature as well as the age of the callus tissue.     

Isolation,Culture and Callus Formation of lpomoea batatas Protoplasts

Numerous viable protoplasts from stem callus cells of Ipomoea batatas tissue culture have been isolated by enzyme treatment involving cellulase EA3 867 (2.0%), CaC12·2H2O (20 mM) and mineral constituent of medium A at pH5.4 in 0.8 M mannitol in 5 hours at 25±1℃. The protoplasts were cultured at a density of 1-2 × 105/ml in solid agar medium E supplemented with 2, 4-D (0.1mg/l) and kinetin (0.1 mg/l), or NAA (0.3 mg/l) and kinetin (0.1 mg/l) in petri dishes, and placed in a controlled growth cabinet maintained at 27 ℃, and illuminated with floureseent light. They regenerated new cell wails after 7 days of culture. The first cell division was observed after 10 days. Ceil division continued thereafter, and after 40 days of culture small white calli (size about 0.2–0.3 mm) were visible in the petri dishes small calli were inoculated in the same nutrients as the protoplasts culture media, but without mannitol. They developed into large calli.     

虎杖愈伤组织的诱导及高产白藜芦醇材料的筛选

将虎杖(Polygonum cuspidatum)不同外植体经不同消毒时间处理后,接种在添加不同激素种类和水平的相同基本培养基上或相同激素种类和水平基本培养基上进行诱导实验,同时对根、根茎芽、叶、韧皮诱导的愈伤组织进行白藜芦醇的含量测定实验,其结果表明:基本培养基以MS较好,外植体叶对激素种类较为敏感,其中适当浓度的NAA诱导愈伤组织比2,4-D的效果要好,KT比BA要好,在有KT存在的培养基上诱导愈伤组织比较紧密,有利于分化;在MS NAA2m g/L KT0.1mg/L培养基上诱导愈伤组织较好,根茎芽的诱导率最高,为73%.愈伤组织的生长趋势从接种的第3d开始生长,到21d时生长达到最高峰,干重为0.461g,以后生长速度减慢.不同材料诱导的愈伤组织中白藜芦醇的含量以根茎部芽的愈伤组织含白藜芦醇最高,其次是叶和根,最低的为韧皮.     

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.     

Plant regeneration via somatic embryogenesis in sugarcane

Plants, regenerated from callus cultures of sugarcane (Saccharum officinarum L.) clone IJ76-316, originated through somatic embryogenesis. Callus cultures were established from primordial leaves and apical meristems on Murashige and Skoog medium (MS) supplemented with 3 mg 1^?1 2,4-dichlorophenoxy acetic acid and 100 ml 1^?1 coconut water (MSC₃). Nodular calli formed within 2 weeks of culture. Calli were maintained on MSC₃ medium by transfer every 3 to 4 weeks. Somatic embryogenesis occurred after 10 weeks culture of callus on MSC₃ medium. Somatic embryogenesis was also observed in cell suspension cultures initiated from calli maintained on MSC₃ and then cultured in half strength MS liquid medium supplemented with 0.5 mg 1^?1 2,4-D. Somatic embryos produced coleoptiles and shoots 2 to 4 weeks after transfer to MS medium supplemented with 100 ml 1^?1 coconut water (MSC), and produced complete plantlets within 4 weeks of further culture on half-strengh MS medium (half-MS) with 30 g 1^?1 sucrose. Calli grown on MSC₃ medium, when transferred to half-MS medium containing 15 g 1^?1 sucrose, produced tiny plantlets, circa 4–10 mm, without forming coleoptiles, suggesting precocious germination of somatic embryos. The regenerates included morphological variants.     

Growth, Water Content, and Proline Accumulation in Drought-Stressed Callus of Date Palm

This study was conducted to examine the response of date palm (Phoenix dactylifera L., cvs. Barhee and Hillali) calli to water stress. Callus derived from shoot tip explants was inoculated in liquid Murashige and Skoog medium containing 10 mg dm^–3-naphthaleneacetic acid, 1.5 mg dm^–3 2-isopentenyladenine, and 0 to 30 % (m/v) polyethylene glycol (PEG 8000) to examine the effect of water stress. After 2 weeks, callus growth, water content, and proline accumulation were assessed. Increasing water stress caused a progressive reduction in growth as expressed in callus fresh mass, relative growth rate, and index of tolerance. Both genotypes tested followed this general trend, however, cv. Barhee was more tolerant to drought stress than cv. Hillali. Increasing PEG concentration was also associated with a progressive reduction in water content and increased content of endogenous free proline.     

Changes in the Phytohormone Levels in Wheat Calli as Affected by Salicylic Acid and Infection with Tilletia caries,a Bunt Pathogenic Agent

The role of salicylic acid (SA) in growth regulation and the change in the levels of phytohormones (IAA, ABA, and cytokinins) were studied in the wheat calli co-cultured with bunt pathogen Tilletia caries. Calli infection with T. caries resulted in the hypertrophied callus growth and simultaneous increase in phytohormone level. The addition of SA to the nutrient media decreased the callus growth induced by the pathogen, whereas the level of investigated phytohormones was not affected. In the SA-treated infected calli, the formation of necrotic lesions was observed in the zones of contact of the fungal mycelium with callus cells that limited pathogen growth. The authors suggest that the stabilization of the hormonal balance of plant cells at pathogenesis is one of the possible mechanisms of the SA protective action in vitro and in vivo. Hence, co-culturing wheat calli and T. caries fungus appeared to be a convenient model for assessing SA protective action.     

Growth characteristic and ion contents of rice callus under the influence of NaCl and hydroxyproline

Calli of salt tolerant (Bhoora rata) and salt susceptible (GR₁₁) rice varieties were cultured on Linsmaeir and Skoog’s medium containing LD₅₀ concentration of NaCl (200 mM) and hydroxyproline (10 mM). Growth rate of callus and Na⁺, K⁺, Cl⁻, Mg⁺², and Ca⁺² contents of the cultured rice tissues were determined at the end of 0, 2, 4 and 6 weeks of incubation. Hydroxyproline resistant calli of both rice varieties when cultured on Linsmaeir and Skoog’s medium containing both NaCl and hydroxyproline showed increased dry weight and enhanced intracellular levels of K⁺, Mg⁺² and Ca⁺². The accumulation of Na⁺ and Cl⁻ ions was less in the hydroxyproline resistant calli.     

Comparison of 2,4-D and picloram for selection of long-term totipotent green callus cultures of sugarcane

Long-term regeneration of sugarcane (Saccharum spp. hybrid and Saccharum spontaneum L.) callus cultures was achieved by selection of green callus on MS agar medium containing 0.5 mgl^-1 picloram or 2,4-D. Newly initiated sugarcane callus cultures were a complex mixture of different tissue types including white, nonregenerative and green, regenerative tissues. The proportion of the tissue types changed as a function of time in culture, genotype, and amount and kind of auxin. Green callus on picloram media always regenerated green plants. Nine hybrids and ten wild relatives of sugarcane produced green calli on picloram media whereas only three hybrids were grown as green calli on 2,4-D media in long-term culture. Green calli were inoculated into liquid MS medium with 0.5 mgl^-1 picloram for suspension culture. These cultures were totipotent after 19 months. For routine culture, we initiated callus cultures on modified MS medium with 3 mgl^-1 2,4-D, then in two to three weeks we subcultured callus on MS medium with 0.5 mgl^-1 picloram and selected for green callus. Green calli regenerated large numbers of green plants after more than four years.