Condensed tannins in the tissue culture of sainfoin (Onobrychis viciifolia Scop.) and birdsfoot trefoil (Lotus corniculatus L.)

Two forage legumes, birdsfoot trefoil (Lotus corniculatus L.) and sainfoin (Onobrychis viciifolia Scop.), containing condensed tannins in their leaves and stems were used as source material to study condensed tannins in tissue culture. More protoplasts were isolated from mesophyll tissue of a low tannin-containing strain of birdsfoot trefoil than from a high tannin-containing strain, but more tannin-filled protoplasts were observed in the latter. Growth rates of leaf explant-derived callus tissue were greater for the high-tannin than for the low-tannin strain. In sainfoin, callus cultures from leaf explants produced numerous tannin-filled cells by 21 days. Explants from sainfoin cotyledons and roots, tissues which normally do not contain tannins, also formed callus with tannin-filled cells in 21 days but in almost every case, a cytokinin was required for tannin formation to occur. The occurrence of tannin-filled cells in callus from root and cotyledon explants was variable and genotype specific. These results show that endogenous tannins can affect protoplast isolation and possibly callus growth in birds-foot trefoil, and that the formation of condensed tannins in sainfoin callus culture can be influenced by a growth regulator.Abbreviations BAP benzylaminopurine - KIN kinetin - NAA naphthaleneacetic acid - PAR photosynthetically active radiation Contribution no. 920 of Agriculture Canada Research Station, 107 Science Cres., Saskatoon, Saskatchewan, Canada S7N OX2     

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl₂.2H₂O produced an average of 4.5 × 10⁶ protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10⁵ protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - FDA fluorescein diacetate NRCC No. 27937     

Regeneration of plantlets from cell suspension culture derived callus of white poplar (Populus alba L.)

Friable calli derived from the stem tissues of Populus alba were used to establish cell suspension cultures which were characterized for in vitro growth and regeneration capacity. Suspended cells and callus recovered from these cells were maximal on a fresh weight basis using MS liquid medium containing 0.44 M BAP and 4.52 M 2,4-D. Shoot regeneration from the recovered callus was observed within 30 to 40 days of culture. The number of shoots was increased by subculturing the shoot-forming callus 2 to 3 times on MS medium supplemented with 19.7 M 2iP and 0.05 M IBA. Regenerated shoots were easily rooted on half-strength MS medium lacking growth regulators, and the plantlets were transferred to pots containing vermiculite for greenhouse growth.Abbreviations BAP 6-benzylaminopurine - 2iP 2-isopentenyladenine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - MS medium Murashige and Skoog medium (1962)     

翅果油树茎段愈伤组织和芽发生的组织学研究

本文对翅果油树大宫灯型茎段培养在ＭＳ附加６－ＢＡ较高、ＮＡＡ较低浓度的培养基上培养０～３０ｄ的组织学变化进行了研究。创伤对其愈伤组织的形成有明显的刺激作用,培养３～４ｄ切口处的皮层细胞、形成层细胞、韧皮部薄壁细胞以及髓组织细胞,甚至表皮细胞均脱分化开始分裂;培养８～１１ｄ,切口明显膨大,起源于髓及维管组织周围薄壁细胞的愈伤组织突起大;培养１２～２０ｄ愈伤组织块中出现了分生组织和维管组织结节;培养２１～３０ｄ,愈伤组织表层和近表层细胞分化出芽原基,但与维管组织结节无直接联系。     

Induced Caulogenesis in Long-term Callus Cultures of Rosrnarinus officinalis L

The callus formed in Rosmarinus officinalis L in association with shoot tip proliferation was isolated and subjected to different treatments for good growth. Two basal media, namely, Murashige and Skoog (MS) and Schenk and Hildebrdndt (SH) and their modifications supplemented with 0.25 mg I^-1 6-benzylaminopurine (BAP), 0.5 mg I^-1 indole-3-acetic acid (IAA) and 1.0 mg I^-1 2,4-dichlorophenoxyacetic acid (2,4-D) were used. Callus in MS medium, was compact and remained fresh and green upto 30 days but grew slowly. Whereas, in SH medium callus growth was rapid but it turned brown within 15 days.The browning of callus could be checked with the addition of 1500 mg I^-1 NH,NO, to the medium, in which callus grew 15 fold in fresh weight during 21 days and remained fresh upto 45 days of incubation.The shoot buds differentiated in this somatic callus with the addition of 0.5 mg I^-1 each of BAP, 2-isopentenyl adenine (2ip), IAA and 10 mg I^-1 gibberellic acid (GA₃), within 15 days of incubation provided the callus remained floating on the liquid medium. Histological investigations revealed both peripheral and occasionally internal differentiation of shoot buds. Differentiated shoot buds were proliferated, rooted and transplanted in the soil.     

Long-term optimized embryogenic cultures in durum wheat (Triticum durum Desf.)

Five varieties of durum wheat: Appulo, Ofanto, Latino, Creso, and Castello (Triticum durum Desf.) adapted to the semi-arid mediterranean environment have been tested for their in vitro response. Compact, embryogenic, highly regenerable calli originated from primary callus derived through proliferation of the scutellum of immature embryos explanted in the presence of 2,4 dichlorophenoxyacetic acid. Selective subculture of the white, compact, embryogenic sectors led to the establishment of long-term cultures. Regeneration occurred on hormone-free medium either via germination of somatic embryos, or via multiple-shoot formation probably due to precocious germination of somatic embryos. The three varieties, Ofanto, Creso and Appulo, were the best responding genotypes. Callus fragmentation and two subsequent transfers onto fresh medium at 7-day intervals yielded a frequency of plant regeneration of some 25–40 plantlets per gram fresh weight callus in 21 days on Murashige and Skoog's hormone-free medium. Plantlets could be efficiently established in soil, thus confirming the possibility of biotechnological approaches with varieties of this crop species.Abbreviations E embryogenic - NE non embryogenic - MS Murashige and Skoog's (1962) medium - 2,4-D 2,4 dichlorophenoxyacetic acid - DAA days after anthesis - FWT fresh weight tissue     

Plant regeneration from mesophyll protoplasts of Centaurea cyanus,Senecio x hybridus and Callistephus chinensis

Protoplasts were isolated from leaves of glasshouse-grown plants of Centaurea cyanus and axenic shoot cultures of Senecio x hybridus. Upon culture, using modified MS-based media, protoplasts of both systems entered division to produce callus, followed by plant regeneration. Leaf protoplasts of Callistephus chinensis entered sustained division only following the preconditioning for 24h of peeled leaf tissues on agar-solidified MS-based medium. Protoplasts were also isolated from cell suspensions of C. chinensis and divided in MS-based or KM media. However, only leaf mesophyll protoplasts of Callistephus produced callus, which developed shoots.The establishment of protoplast-to-plant protocols for these ornamental species has provided a basis for broadening their gene pools through somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - KM Kao and Michayluk (1975) - g.f.wt. gram fresh weight     

Semi-continuous production of sanguinarine and dihydrosanguinarine by Papaver somniferum L. cell suspension cultures treated with fungal homogenate

Papaver somniferum L. (opium poppy) cells were elicited with a Botrytis sp. homogenate and cultured by a semi-continuous process. Elicitation induced synthesis of sanguinarine and dihydrosanguinarine. Significant release of both alkaloids into the culture medium occurred. Medium exchange at 2-day intervals enabled product recovery from spent medium and maintained culture viability. Culture growth was not inhibited by elicitor treatment necessitating sub-culture prior to re-elicitation. Re-elicited cultures displayed an increasing sensitivity (reduced growth rate, higher alkaloid yield) to the elicitor with each successive treatment and did not survive a fourth elicitation.Abbreviations DW dry weight - FW fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - SGE sanguinarine - DSGE dihydrosanguinarine Publication 29143 from the National Research Council of Canada     

Induction of division and differentiation of somatic embryos in the leaf epidermis of Gaillardia picta

Somatic embroys and subsequent plant regeneration were obtained from isolated leaf epidermis of Gaillardia picta. Abaxial and adaxial epidermal peels (monolayer) from 45 days old aseptic seedlings were isolated and segments measuring 5 mm x 3 mm were cultured on B₅ basal medium supplemented with various growth regulators such as naphthaleneacetic acid or indolebutyric acid and benzylaminopurine or kinetin. Within 12 h of culture the epidermal cells showed receding of cytoplasm from the walls. After 48 h of incubation 3 or 4 localized zones, each consisting of 3–8 cells that accumulated cytoplasm and stained densely, were observed. Mitotic divisions occurred in these zones on day 3 of culture and localized masses of callus were observed in 95% of the cultures after 10 days. In another 5 days, the callus differentiated somatic embryos or roots, depending on the growth regulators and their concentration in the medium.Abbreviations Used BAP 6-benzylamiopurine - IBA indolebutyric acid - Kn kinetin - NAA -naphthaleneacetic acid     

Callus induction and high frequency plant regeneration in Italian millet (Setaria italica)

Callus was induced from mature seeds of two cultivars of Setaria italica (L.) on Murashige and Skoog's medium (1962) supplemented with 2mg/l 2,4-D and 0.5 mg/l KN. Regenerating ability of the callus was better in the cultivar 315 compared to 212. Organogenesis was influenced not only by cytokinin, but also by the sucrose concentration in the medium. High frequency (80%) plant regeneration was achieved and quantified on the basis of callus fresh weight. The ability of the callus (cultivar 212) to regenerate whole plants was retained until the 5th passage, but during the 6th passage it declined considerably.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KN Kinetin - BAP 6-benzylaminopurine - MS Murashige and Skoog basal medium     

罗汉果组织培养中愈伤组织和腋生枝的形成

用罗汉果茎段为外植体,培养在MS+BA1.0+IBA0.1毫克/升培养基上,诱导愈伤组织和芽形成。观察了罗汉果茎段愈伤组织的生长以及腋生枝的形成。罗汉果茎段培养5天后,潜伏腋芽开始萌动和生长。培养10天后罗汉果茎段的基部一端开始膨大。培养20天后产生大量白色疏松的愈伤组织,这时腋生枝已经长成3—6厘米长。培养30天后基部的愈伤组织中有少量瘤状小突起,但再分化形成芽的频率极低。结果表明,罗汉果茎段组培形成的苗均是从腋芽产生的。     

Embryogenic culture initiation and somatic embryo development in hybrid firs (Abies alba×Abies cephalonica,and Abies alba×Abies numidica)

Summary Embryogenic cultures were established from silver fir (Abies alba Mill.) female megagametliophytes with developing embryos and from excised mature embryos after pollination with Abies cephalonica Lond. or Abies numidica DeLann pollea The frequency of embryogenic callus formation was dependent on genotype, collection time, medium and explants used. The embryogenic callus initiation potential of megagamethophytes with developing embryos in both hybrids was higher in early July and dropped as the zygotic embryos matured. Excised cotyledonary embryos were less suitable for induction of embryogenic cultures. SH medium supplemented with 1mg/l BAP was the most efficient for callus induction and maintenance. Cultures were composed of early somatic embryos with an embryonal mass formed of highly cytoplasmic cells, rich in cell organelles and a suspensor built up by vacuolated, strongly elongated cells. Maturation of embryos was detected with the formation of bipolar structures with shoot and root apices. Nutrition reserves were observed in cells of embryos cultured on DCR medium containing 1 or 10 mg/l ABA. Cotyledon formation, hypocotyl elongation and low frequency germination occured following transfer of the embryos to the same medium without ABA.     

Sugarcane micropropagation and phenolic excretion

Sugarcane shoot formation was followed using a temporary immersion system. Plant fresh weight, plant dry weight, shoot number and phenolic excretion to the culture medium were recorded during shoot formation. Shoot number increased for 30 days of culture but formation of new shoots was greatly reduced from 31 to 40 days. Phenolic excretion also increased during the first 20 days of culture (gallic acid represented 82% total phenolics) and decreased during the last 10 days (31–40 days of culture). The most intensive period of phenolic excretion (11–20 days) preceded the most intensive period of shoot formation (21–30 days). The same relationship does not seem to exist between the accumulation of fresh and dry weights. Subculture onto fresh medium at the beginning of proliferation (10 days after culture initiation) was detrimental to shoot formation in the subsequent period (11–20 days). However, such a detrimental effect could be avoided if gallic acid was added to the medium. Addition of cysteine to the culture medium reduced both excretion of phenolics and shoot formation but not fresh weight. The use of temporary immersion systems, the increase of culture medium volume per initial explant and the addition of paclobutrazol promoted both phenolic excretion and sugarcane shoot formation. Results presented here indicate a relationship between phenolic excretion and shoot formation but not with accumulation of plant weight. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from protoplasts of Felicia and Brachycome

Protoplasts were isolated from leaves of axenic shoot cultures of Felicia bergeriana (Kingfisher Daisy) and Brachycome iberidifolia (Swan River Daisy) and from callus cultures of Felicia. Plants were regenerated from all three sources and since both species are of ornamental value (blue flowered) the establishment of plant regeneration provides a basis for their incorporation in somatic hybridisation programmes involving important ornamentals such as Chrysanthemum.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - KIN 6-furfurylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog (1962) - FDA fluorescein diacetate - f. wt. fresh weight     

Effect of Brassinolide on Growth and Shikonin Formation in Cultured Onosma paniculatum Cells

The effect of brassinolide (BR) on cell growth and shikonin and its derivative formation in Onosma paniculatum cell culture was studied. BR addition with IAA and BAP (+BR/+IAA/+BAP) in B₅ medium slightly increased the cell growth at 0.01–0.1 ppb concentration compared with a growth control (−BR/+IAA/+BAP). Only BR addition (+BR/−IAA/−BAP) at 0.001–100 ppb in B₅ medium significantly increased the cell fresh weight compared with a growth control (−BR/−IAA/−BAP). The same concentration of BR tested at 0–1000 ppb increased the cell fresh weight of +IAA/+BAP significantly more than that of −IAA/−BAP. BR at 0.001–0.1 ppb with IAA and BAP added (+BR/+IAA/+BAP) in M₉ medium increased shikonin and its derivative content markedly by 31–87%, compared with its control (−BR/+IAA/+BAP). BR at 0.001–1000 ppb without IAA and BAP added to M₉ medium (+BR/−IAA/−BAP) also increased shikonin and its derivative content compared with its control (−BR/−IAA/−BAP). However, the amount of shikonin and derivative formed of +IAA/+BAP was greater than that of −IAA/−BAP only at the same concentration of BR at 0–1 ppb. These combined results show that BR at 0.01 ppb with IAA and BAP added was the best for cell growth and shikonin formation. Formation of shikonin and its derivative by adding BR at 0.01 ppb with IAA and BAP (+BR/+IAA/+BAP) in M₉ medium was significantly enhanced 4 days after BR addition compared with a production control (−BR/+IAA/+BAP). In contrast, +BR/−IAA/−BAP vs. −BR/−IAA/−BAP was not as effective as +BR/+IAA/+BAP vs. −BR/+IAA/+BAP for the shikonin formation. The time course study for shikonin formation also showed that +BR/+IAA/+BAP and −BP/+IAA/+BAP only slightly increased cell growth in M₉ medium. Similarly, soluble protein content in the cells treated by BR at 0.01 ppb with IAA and BAP (+BR/+IAA/+BAP) exceeded that of the control (−BR/+IAA/+BAP) 4 days after BR addition. And +BR/−IAA/−BAP only slightly increased the soluble protein content over that of −BR/−IAA/−BAP. Received November 2, 1998; accepted August 25, 1999     

Regeneration of plants from callus tissue of the pasture legume Centrosema brasilianum

Plants were regenerated from leaf explants of Centrosema brasilianum cultured in vitro. Callus and buds were produced on Murashige and Skoog medium (MS), 0.8% agar, 0.1 mg/l NAA and 1 mg/l BAP. Regeneration of multiple shoots was achieved by transferring callus onto fresh medium containing 0.01 and 1 mg/l of NAA and BAP, respectively. Shoots formed roots upon transfer to MS with 0.01 mg/l NAA. Plantlets were succesfully transferred to soil. Leaf-derived calli of Centrosema arenarium, C. macrocarpum, C. pascuorum, C. pubescens, and C. virginianum did not produce shoots when cultured in vitro.     

卫星搭载红豆草后代中耐盐细胞系的筛选及鉴定

将红豆草种子搭载于940703返地卫星,经田间繁殖得后代种子,先将种子在1.5%NaCl上筛选、并在该盐浓度下诱导愈伤组织和筛选,在无盐培养基上恢复生长后再在1.2%NaCl上筛选得到耐盐变异系.变异系具有正常的分化能力并表现出对PEG胁迫的交叉抗性.变异系在无胁迫条件下脯氨酸含量较低但在有盐胁迫时具有高效积累脯氨酸的能力.后者可能对红豆草耐盐系更为重要.变异系中脯氨酸的这种合成机理可能是由于一些基因在调控中对水的敏感性改变引起.梯度聚丙烯酰胺凝胶电泳表明耐盐系的SOD和酯酶分别出现175kD和75kD的新形式.说明空间诱变和组织培养相结合可以筛选耐盐变异系.     

Induction of somatic embryogenesis in leaf-derived callus of Vicia narbonensis L.

A method for the induction of somatic embryogenesis in callus cultures, using explants from mature leaves of Vicia narbonensis L., is described. Callus developed on a solid medium (Murashige and Skoog 1962), which was supplemented with low concentrations of picloram and benzylaminopurine. Subsequent culture was carried out in different liquid media (culture length four months). The gradual reduction of auxin and cytokinin concentrations, and the addition of glutamine and pyridoxal·HCl were favourable. Somatic embryos appeared on solid media without phytohormones.Abbreviations MS Murashige and Skoog (1962) - 2,4 D dichlorophenoxy acetic acid - KIN 6-furfurylaminopurine - BAP 6-benzylaminopurine - picloram 4-amino-3,5,6-trichloropicolinic acid - NAA 1-naphthalene acetic acid - p-CPA parachlorophenoxy acetic acid - M1 - M7 media numbers (details in materials and methods)     

Morphogenesis and plant regeneration from cultured endosperm of Emblica officinalis Gaertn

Mature endosperm of Emblica Officinalis (Euphorbiaceae) formed a continously growing callus on MS medium supplemented with an auxin (2,4-D or IAA) and a cytokinin (K or BAP). Subculture of callus on MS with BAP (0.2 mg/l) and IAA (0.1 mg/l) resulted in formation of shoots and embryo-like structures in 50 and 8 per cent cultures, respectively. Regeneration of shoots was more frequent when both BAP (0.2 mg/l) and IAA (0.1 mg/l) were present than on BAP (0.2 mg/l) alone. The embryo-like structures produced plantlets.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indoleacetic acid - K kinetin - NAA naphthaleneacetic acid - PDB para-dichlorobenzene (née Arora)