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.     

Peroxidase and catalase changes during in vitro adventitious shoot organogenesis from hypocotyls of Albizia odoratissima L.f. (Benth)

Hypocotyls of Albizia odoratissima cultured on shoot induction medium (MS medium with 7.5 μM BAP and 0.5 μM NAA) showed adventitious shoot organogenesis under light with 16 h photoperiod. Similar cultures under total darkness produced non-morphogenic calli. The changes in the specific peroxidase and catalase activity, total protein content and acidic isoperoxidase pattern were compared between the culture showing shoot organogenesis and culture producing non-morphogenic calli. It was found that in vitro shoot bud differentiation is accompanied by an increase of the specific activities of peroxidase and catalase in culture kept under light. In parallel with the above changes the total protein content reached to the maximum level and also a new isoperoxidase (P10) expressed on the 21st day in cultures kept under light. Conversely, culture producing non-morphogenic calli underwent a reverse change in specific peroxidase activity. This change in antioxidant enzyme activities corresponds to the histological observation of shoot bud differentiation in cultures kept under light.     

Histology of somatic embryogenesis in cultured leaf segments of sugarcane plantlets

Calli have been initiated from young leaves of in vitro grown sugarcane shoots. Histological examination has shown that the two types of calli induced (nodular and friable) originated from different regions of the explants and were cytologically different.This study has shown an obvious relation between the developmental stage of the excised tissue and the potential of plant regeneration of the in vitro initiated callus culture. Nodular calli were obtained from bases of the fast-growing young leaves while their more mature parts of the older leaves only produced friable calli. High-frequency plant regeneration via somatic embryogenesis was obtained from nodular calli while friable calli rarely produced plantlets.     

Morphological differentiation and occurrence of thiophenes in leaf callus cultures from Tagetes species: Relation to the growth medium of the plants

The morphology, and the number and concentration of thiophene-like compounds were studied in leaves, roots and calli of Tagetes species grown with different nutrient solutions. The type of nutrient solution exerted no effect on the number of thiophene-like compounds, but altered the type of morphological differentiation and thiophene content of calli. Calli of T. minuta L. showed little differentiation and resulted in suspensible callus after two passages. Calli of T. erecta L. cv. Rose d'Inde differentiated rapidly and turned dark brown after one passage. The morphology of calli from T. patula L. cv. Nana furia was intermediate. Tertiary callus of T. patula contained more thiophene-like compounds and higher concentrations than did the corresponding calli of T. minuta . The content of thiophene-like compounds decreased after various callus passages, but the relative decrease varied between species.     

Biochemical parameters to assess cell differentiation of Bouvardia ternifolia Schlecht callus

Calli derived from leaves and radicles of B. ternifolia were grown on Murashige and Skoog (MS) basal medium, and the effects of different nitrogen sources on the rate of callus growth and on the enzymes related to nitrogen assimilation were studied. Ammonium alone did not support callus growth unless a Krebs-cycle intermediate was added to the medium. The activities of glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.7.1), and glutamate dehydrogenase (EC 1.4.1.2) were measured in homogenates of callus grown on media supplied with different nitrogen sources. The results indicate that leaf and root calli have similar levels of these enzymes when grown on MS medium (Murashige and Skoog 1962. Physiol. Plant. 15, 473–497). However, when the calli were supplied with glutamine as the sole nitrogen source, the activity of glutamate synthase increased in leaf callus but was almost completely inhibited in root callus. The results indicate that calli originated from different B. ternifolia tissues do not have the same biochemical dedifferentiated state.     

Accumulation of a 31-kDa glycoprotein in association with the expression of embryogenic potential by spinach callus in culture

Calli grown from segments of spinach ( Spinacia oleracea L.) root in the presence of gibberellic acid (GA₃) plus auxin, differentiated to yield somatic embryos after transfer to a medium without growth regulators, while calli formed in the absence of GA₃ failed to generate any embryos. We extracted proteins from the two types of callus and analysed them by polyacrylamide gel electrophoresis. Compared with the proteins from calli formed on medium that contained only naphthaleneacetic acid (NAA) as a growth regulator, the proteins from calli grown in the presence of GA₃ included appreciably higher levels of a 31-kDa basic protein (pI = 8.8). The protein resembled type I ribosome-inactivating proteins (EC 3.2.2.22) in terms of molecular mass, isoelectric point, sequence of amino-terminal amino acids and extent of glycosylation. The 31-kDa protein was barely detectable in extracts of various tissues from seedlings. Thus, it is possible that an increase in the relative level of this protein might be associated with the expression of embryogenic potential expressed by spinach callus.     

In situ and in vitro senescence induced by KCl stress: nutritional imbalance, lipid peroxidation and antioxidant metabolism

Sunflower (Helianthus annuus L. cv. SH222) plants and calli were exposed to KCl stress for three weeks. Calli were more tolerant to KCl than plants. KCl stress decreased NO(-)(3), Mn, Fe and B levels in whole plants and P, Ca and Mg in shoots. NO(-)(3), P, Ca, Mg, Mn, and B levels decreased in 100 mM-stressed calli. Chlorophyll content, F:(m) and (F:(m)-F:(0))/F:(m) ratio decreased in stressed leaves, while F:(0) increased only in leaves exposed to severe stress (100 and 150 mM). Membrane permeability and lipid peroxidation increased in plants under all stress conditions and in 100 and 150 mM stressed calli, but remained unchanged in 25 mM stressed calli. Salt stress also induced changes relating to antioxidant enzymes: plants under all stress conditions showed a decrease in catalase, peroxidase and SOD activities. Calli under moderate stress (25 mM KCl) showed an increase of catalase, peroxidase and SOD activities, but the activities of peroxidase and SOD decreased when calli were exposed to higher KCl concentrations. The decrease of antioxidant enzyme activities is in tune with lipid peroxidation and membrane permeability increases. On the other hand, calli adapted for 6 months to 100 mM KCl showed an increase of these enzyme activities compared to unstressed calli, while MDA production and membrane permeability were not significantly affected.     

油樟悬浮培养及次生代谢产物的诱导

以油樟(Cinnamomum longepaniculatum)叶片为外植体在附加6-BA和NAA不同激素浓度组合的MS培养基上筛选质地疏松、生长旺盛的愈伤组织, 分别接种在MS、B5、WPM三种液体培养基中进行细胞悬浮培养, 并检测诱导产生的次生代谢产物。结果表明: 2 mg.L-1 6-BA+ 0.5 mg.L-1 NAA能诱导质地疏松、生长旺盛的愈伤组织, B5基本培养基中细胞长势最好; 愈伤组织在B5+2 mg.L-1 6-BA+ 0.5 mg.L-1 NAA中悬浮培养, 继代2次后形成均一的单细胞; 从油樟悬浮培养物中检测出50%以上的成分是苯甲醇。     

银杏愈伤组织培养及其黄酮类化合物的测定(简报)

采用银杏胚、胚乳及3个月苗龄的苗叶为外植体,在附加不同激素的培养基上诱导出愈伤组织,从愈伤组织中提取黄酮类化合物并测定其含量。结果表明,由胚诱导的愈伤组织中黄酮类化合物含量最高。     

In vitro selection of resistant/tolerant mutants lines of Citrus jambhiri Lush. using crude culture filtrate of Phytophthora parasitica and their randomly amplified polymorphic DNA analysis

This study deals with in vitro‐induced mutagenesis and selection of Phytophthora tolerant lines of Citrus jambhiri and their regeneration. For in vitro‐induced mutagenesis, cotyledons were treated with ethyl methane sulfonate (EMS) 100, 200 and 300 mm for different time durations viz. 1, 3, 6 and 9 hr. Callus cultures were established from EMS treated cotyledon explants on MS medium supplemented with 2.0 mg/L of 2,4‐D and 500 mg/L of malt extract. Calli derived from cotyledon were challenged in vitro on selective MS medium containing 5%–100% of culture filtrate (CF) of the Phytophthora parasitica. Selected mutagen‐treated calli showed resistance in vitro on media containing CF. Calli treated with 100 mm EMS for 6‐hr duration showed tolerance (24%) up to 75% CF after 4th selection cycle. While, calli treated with 200 mm for 6‐hr duration showed maximum tolerance (76%) up to 75% CF. Resistant calli were then transferred to MS regeneration medium for shoot bud regeneration. A dose‐dependent decrease in the regeneration capacity of the selected calli was observed with the increasing concentration of the CF. In randomly amplified polymorphic DNA analysis, plantlets showed different banding pattern in comparison with the control plant, which confirms the presence of variations at genetic level.     

油樟悬浮培养及次生代谢产物的诱导

以油樟（Cinnamomum longepaniculatum）叶片为外植体在附加6-BA和NAA不同激素浓度组合的MS培养基上筛选质地疏松、生长旺盛的愈伤组织,分别接种在MS、B5、WPM三种液体培养基中进行细胞悬浮培养,并检测诱导产生的次生代谢产物。结果表明：2mg·L^-16-BA＋0.5mg·L^-1NAA能诱导质地疏松、生长旺盛的愈伤组织,B5基本培养基中细胞长势最好;愈伤组织在B5＋2mg·L^-16-BA＋0.5mg·L^-1NAA中悬浮培养,继代2次后形成均一的单细胞;从油樟悬浮培养物中检测出50%以上的成分是苯甲醇。     

Tolerance to NaCl induces changes in plasma membrane lipid composition,fluidity and H+-ATPase activity of tomato calli

Two tomato ( Lycopersicon esculentum Mill. cv. Pera) callus lines tolerant to NaCl were obtained by successive subcultures of NaCl-sensitive calli in 50 and 100 m M NaCl-supplemented medium. Growth and ion content, as well as plasma membrane lipid composition, fluidity and H⁺-ATPase (EC 3.6.1.35) activity, were studied in both NaCl-sensitive and NaCl-tolerant calli. Although calli tolerant to 100 m M NaCl exhibited a reduced growth relative to calli sensitive to NaCl or tolerant to 50 m M NaCl, growth of calli tolerant to 100 m M NaCl was higher than that of NaCl-sensitive calli grown for one subculture in 100 m M NaCl. Growth in the presence of 100 m M NaCl provoked an increase of Na⁺ and Cl⁻ content, but no significant changes in K⁺ and Ca²⁺. As compared with NaCl-sensitive and 50 m M NaCl-tolerant calli, plasma membrane vesicles isolated from calli tolerant to 100 m M NaCl exhibited a higher phospholipid and sterol content as well as a lower phospholipid/free sterol ratio and a lower double bond index (DBI) of phospholipid fatty acids. The changes in plasma membrane lipid composition were correlated with a decrease of plasma membrane fluidity in calli tolerant to 100 m M NaCl, as indicated by fluorimetric studies using diphenylhexatriene (DPH) as probe. Plasma membrane-enriched vesicles isolated from calli tolerant to 100 m M NaCl showed lower ATP hydrolysis and ATP-dependent H⁺-pumping activities, as well as a lower passive permeability to H⁺ than plasma membrane from NaCl-sensitive and 50 m M NaCl-tolerant calli. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H⁺-ATPase activity in salt tolerance of tomato calli is discussed.     

杯山药零余子愈伤组织诱导及植株再生的研究

对怀山药（Ｄｉｏｓｃｏｒｅａ ｏｐｐｏｓｉｔａ）零余子愈伤组织的诱导、分化、再生苗的生根和移栽进行了研究。结果表明：⑴在不同激素组合的培养基上怀山药零余子均能产生愈作组织,而且具有一次成苗的能力。ＢＡ２ｍｇ／Ｌ＋ＮＡＡ２ｍｇ／Ｌ的培养基对诱导愈伤组织最有利,其出愈率达１００％;⑵在愈伤组织的分化中,ＢＡ１ｍｇ／Ｌ＋ＮＡＡ１ｍｇ／Ｌ的激素组合是最佳的,其分化率为６３．６％,且多形成丛生芽;⑶再生植株     

Effect of culture medium and light conditions on the morphological characteristics and carbohydrate contents of Medicago strasseri calli

Using 6 culture media (12, 12D, 12G, 11, A and B) made up of MS medium (Murashige-Skoog, 1962) supplemented or not with glycerine, with different cytokinins, and/or 2,4-D, the morphological characteristics and contents in total carbohydrates, reducing sugars, sucrose and starch were studied in calli induced from explants (cotyledon, petiole, hypocotyl and leaf) obtained from Medicago strasseri seedlings. Callus formation was induced under photoperiod (16h light/8h darkness) conditions or in the absence of light. Considerable variability in the calli was observed, depending on the explants and media used. Under photoperiod conditions, medium A with KIN (1 mg/l) and 2,4-D (3 mg/l) induced many calli with the highest contents in total carbohydrates (886.1–889.3 mg/g DW), sucrose (132.1–188.2 mg/g DW) and starch (125.2–247.6 mg/g DW) and the lowest contents in reducing sugars (118.4–173.3 mg/g DW). In media 11, A and B, under conditions of darkness, calli degenerated at the start of culture. Calli developed in darkness generally had dry weights and total carbohydrate and starch contents lower than those cultured under photoperiod conditions. However, sucrose contents were greater in calli formed in darkness. At these cultures times, differentiation, in the form of organogenesis, was only seen using medium B with cotyledons, petioles and leaves as explants. It was also observed when petioles were cultured in medium A but with a less pronounced organogenic response.     

Determination of phenolic antioxidant compounds produced by calli and cell suspensions of sage (Salvia officinalis L.)

Sage (Salvia officinalis L.) calli were established by culturing internodal segments, excised from aseptic seedlings, on MS basal medium gellied with agar and supplemented with 0.05 mg/L dichlorophenoxyacetic acid (2,4-D) in presence of benzyladenine (BA) or zeatin (ZEA) or kinetin (KIN), at 1.5 mg/L. Suspended cells were established by transferring one callus to 50 mL of liquid MS basal medium devoid of agar and containing the same type of hormonal supplementation used in respective calli growth. The highest growth of calli and suspensions occurred with 1.5 mg/L ZEA. However, with this cytokinin supplementation, as well as with 1.5 mg/L KIN, both in presence of 0.05 mg/L 2,4-D, suspensions differentiated small root shaped structures. Well shaped, majority single cell suspensions were formed under the effect of 0.05 mg/L 2,4-D and 0.5 mg/L KIN. Calli grown with 0.05 mg/L 2,4-D and 1.5 mg/L BA and suspended cells grown with 0.05 mg/L 2,4-D and ZEA or KIN at 1.5 mg/L, or KIN at 0.5 mg/L, were searched for phenolics production. Twelve phenolic compounds were identified in calli: gallic acid, 3-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, caffeic acid, rosmarinic acid, hesperetin, epirosmanol, hispidulin, genkwanin, carnosol, carnosic acid, and methyl carnosate. With the exception for genkwanin and epirosmanol all of these phenolic compounds were also produced by the sage suspension cultures grown in the presence of 1.5 or 0.5 mg/L KIN. Genkwanin was the only phenolic absent in the suspensions grown with 1.5 ZEA. Suspended cells, grown with 0.5 mg/L KIN, and calli cultures showed the highest specific accumulation of the total phenolics, with rosmarinic acid representing 94-97 percnt;.     

Somatic Embryogenesis and Plant Regeneration from Protoplasts of Cowpea (Vigna sinensis)

Immature cotyledons of cowpea (Vigna sinensis Endlo) were used for protoplast isolation. Enzyme solution for protoplast isolation contained 40% cellulase Onozuka R-10,0.30% Macerozyme R-10 and 2% hemicellulase. The purified protoplasts were cultured in Bs,MS or KM8p liquid medium in dark (25℃) at a density of 1 × 105–5 × 105/ml. The protoplasts started cell division in 3–5 days . Sustained cell divisions resulted ill formation of cell clusters and small calli,with cell division frequency reaching 23%–28% in MS medium . Calli of 2 mm in size were transferred onto MSB (MS salts+B5 vitamins) medium with 2 mg/L 2,4-D, 0. 5mg /L BA forfurther growth. Embryogenic calli appeared on this medium. After passage to fresh medium with the same composition, the embryogenic calli were transferred into MSB liquid medium to establish suspension culture. When the suspended calli were transferred back onto MSB agar medium with 0. 1 mg /L IAA, 0.5mg/L KT, 5% mannitol (cultured in light,2000 lx,12h/d), a lot of adventitious roots formed in 7–10 days, and then somatic embryos formed from the protoplast derived calli. But only a few embryoids developed further into the cotyledonary stage ,and the others died at globular, heart-shaped, or torpeto stage . Finally, some cotyledonary embryoids germinated and developed into plantlets or shoots with leaves.     

Effects of CO(2)-Enrichment and of Aminoacetonitrile on Growth and Photosynthesis of Photoautotrophic Calli of Nicotiana plumbaginifolia

Photoautotrophic calli of Nicotiana plumbaginifolia were grown for 3 weeks under two CO₂ concentrations (500 and 20,000 microliters of CO₂ per liter). Calli cultured at high CO₂ exhibited a two-fold higher rate of growth. At CO₂ test levels, these calli were characterized by a lower net photosynthetic capacity than calli cultured at low CO₂. This diminution due to CO₂ adaptation could be ascribed to a 170% stimulation of dark respiration, a 40% decrease in total ribulose-1,5-bisphosphate carboxylase (Rubisco) activity, and also to a feedback inhibition of photosynthesis: high CO₂ grown calli contained about 5.5-fold more sucrose and three-fold less orthophosphate (Pi) than low CO₂ grown calli. Whether the decrease in Rubisco activity is related to the accumulation of sucrose and to the Pi limitation is discussed. Both calli exhibited a Warburg-effect showing the existence of active photorespiration at low CO₂. In calli grown at low CO₂ with 5 millimolar aminoacetonitrile (AAN), an inhibitor of the glycolate pathway, fresh weight decreased by 25% and chlorophyll content by 40%, dark respiration increased by 50% and net CO₂ uptake decreased by about 60% at 340 microliters of CO₂ per liter and 35% at 10,000 microliters of CO₂ per liter. In these calli, glutamine and glutamate contents were half of control calli. In contrast, AAN did not provoke any noticeable effect in calli grown at high CO₂. In photoautotrophic calli, the inhibition of the glycolate pathway by AAN results in severe perturbations in glutamate metabolism and in chlorophyll biosynthesis.     

花生畸胎瘤冠瘿瘤的诱导及胭脂碱合成酶基因的转移

本文报道了致瘤农杆菌(Agrobacterium lume/aciens)的T(?)菌株对分属于五大类型的41个品种的栽培种花生致瘤实验结果。在41个品种中的6个品种上诱发了畸胎瘤。在无激素的MS培养基上诱发冠瘿瘤产生了愈伤组织。瘤组织、愈伤组织,畸胎瘤上幼苗的茎和叶均含有胭脂碱。