Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro

Summary A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as expiants and a combination of BA and NAA in the regeneration media, up to 70% of expiants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL^–1 and NAA concentration of 1mgL^–1. Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the expiants after the initiation of meristematic activity in the cells about 100m from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium — mediated transformation protocols involving cotyledonary petioles.     

Histological analysis in shoot organogenesis from hypocotyl explants of<Emphasis Type="Italic"> Kandelia candel</Emphasis> (Rhizophoraceae)

In vitro culture of hypocotyl explants from Kandelia candel, a common mangrove species, on hormone-free Murashige and Skoog (MS) medium resulted in shoot formation. Since the hypocotyls showed good potential for in vitro shoot multiplication, the process of bud primordium formation was analyzed from a histological viewpoint. A wound periderm first appeared at the top, exposed cut surface of the explants. The wound-induced meristem continued to divide giving rise to suberized cells oriented towards the cut surface. After formation of the suberized cell layers, the meristem and its inner derivatives differentiated into multilayered, uniformly packed parenchyma cells. Bud primordia differentiated from the dense cytoplasmic cells of the wound-induced meristem just beneath the suberized layer near the severed vascular bundles. Each explant produced several visible shoot buds. Furthermore, histological sections revealed that additional bud primordia were present within the explant just underneath the suberized cells and that these bud primordia appeared to be arrested in their development. The fact that additional bud primordia were present within the explant suggests that further manipulation of the explant is helpful to maximize the potential of this system.     

Increased Survival and Differentiation of Frozen Herbaceous Plant Organ Cultures through Cold Treatment

Cold treatment of donor carnation plants (Dianthus caryophyllus L.) at 4 C for 3 days or more resulted in a doubling in the percentage of excised, frozen shoot apices which survived freezing and a 6- to 7-fold increase in the percentage which formed leaf primordia or shoots. The optimal freezing parameters for both survival and differentiation were as follows: size of the shoot apex-two to three sets of leaf primordia; dimethylsulfoxide concentration in the freezing solution-5%; time in dimethylsulfoxide prior to freezing->30 minutes; average cooling rate-≥50 C/minute; initial warming rate-about 1450 C/minute. In general, the cells in the meristematic region of the shoot apex remained viable after freezing while those in the leaf primordia did not. Viability of the meristematic cells appears to be maintained by preventing the growth of intracellular ice crystals formed during rapid cooling by rapidly passing the tissue through the temperature zone in which lethal crystal growth occurs (mechanism of Luyet). Applications of this technique are discussed.     

Micropropagation of horseradish hairy root by means of adventitious shoot primordia

Adventitious shoot primordia were formed on horseradish hairy root cultured in dark. Plantlet formation frequency from the primordia was higher than that from root fragments. Culture for 26 days provided the adventitious shoot primordia, which had the highest potential for plantlet formation (53% explants at 40 days). Benzyladenine supplementation in the dark caused primordium enlargement, but did not increase the number of primordia formed. After adventitious shoot primordia were encapsulated with calcium alginate, kinetin supplementation (2.0–4.0 M) increased the shoot formation frequency (65–80% explants at 20 days) in the light, but also promoted the undesirable formattion of multiple shoots. Supplementation with naphthaleneacetic acid (0.27–5.4 M) in the calcium alginate beads in light enhanced the root emergence from primordia without inhibition of plantlet formation when the encapsulated beads were put on the agar-medium without naphthaleneacetic acid.     

VARIATION IN APPLE CYTOCHIMERAS

Diploid-tetraploid cytochimeras of apple were investigated by cytological examination of buds from branches selected by the characteristics of the fruits, flowers or pollen. Eight types of cytochimeras were identified on the basis of relative size of cells, nuclei and metaphase mitoses in the apical meristem (protomeristem) and mitoses in meristematic primary tissues. There appeared to be five apical layers which contributed to the stem and four which contributed to the leaves and flower parts. Buds of various cytochimeral patterns (designated by a formula giving the ploidy of the apical layers) were found on some bearing trees propagated from known cytochimeral sources. The most frequently associated types were (a) 2–4–2–2–2 and 2–4–4–4–4 (and 2–4–2–4–4 in one cultivar), or (b) 2–2–2–4–4 and 2x. Some sports were uniformly 2–4–4–4–4. The stability of apple cytochimeras under normal conditions appeared greater in some eultivars than in others. Sprouts from severely pruned 2–2–4–4–4 trees were more variable than unpruned branches. Buds of shoots which grew from radiation-damaged buds were more variable than those from non-irradiated buds and included types not yet found by branch selection. Cytochimeral variation was interpreted to be due to layer replacement resulting from infrequent periclinal divisions in apical or axillary meristems, or from wounding of meristems by ionizing radiation.     

Shoot meristem differentiation in shoot primordia culture (SPC) obtained from root primordia culture (RPC) of Solanum lycopersicoides Dun.

We present a unique example of conversion of the morphogenesis type (from rhisogenesis to shoot organogenesis) in in vitro cultures of Solanum lycopersicoides. Liquid shoot primordia cultures (SPC) were obtained from root primordia culture (RPC) on two kinds of MS-based media with BA. The first SMS₈, contained a full set of organic compounds; the second, ²SMS₈, was devoid of organic compounds except sucrose and edamine. Two weeks after passage of RPC onto both the media, disintegration of root primordia and cell aggregates began. After 8 weeks of cultivation on SMS₈ and ²SMS₈ media, SPC aggregates developed from meristematic cells located near the vascular tissue of disintegrating RPC aggregates. Initiation of shoot meristems started from meristematic cells centers which were localised under the surface of the newly formed SPC aggregates. The change in the type of morphogenesis occurred faster on medium SMS₈, but the SPC on medium ²SMS₈ was characterized by more frequent formation of shoots and plants (45% of aggregates, in the case of SPC on medium SMS₈, and 90% in the case of SPC on medium ²SMS₈). This fact was correlated with the structural organization of the meristematic centers. Our results indicate that an RPC system has high morphogenetic potential due to the continual presence of meristematic cells. The change in the type of morphogenesis was followed by a rebuilding of the aggregate structure on the basis of the meristematic cells already existing in RPC, which gave rise to SPC aggregates from which shoot meristems developed.     

Initial proliferation of cortical cells in the formation of root nodules in Pisum sativum L.

Summary Root nodule initiation in Pisum sativum begins with cell divisions in the inner cortex at some distance from the advancing infection thread. After penetrating almost the entire cortex, the branches of the thread infiltrate the meristematic area previously initiated in the inner cortical cells. These cells are soon invaded by bacteria released from the infection thread and subsequently differentiate into non-dividing, bacteriod-containing cells. As the initial meristematic centre in the inner cortex is thus lost to bacteroid formation, new meristematic activity is initiated in neighbouring cortical cells. As development proceeds, more cortical layers contribute to the nodule, with the peripheral layer and apical meristem of the nodule not invaded by bacteria.Lateral root primordia are initiated in a region separate from that in which nodules are formed, with the lateral primordia being closer to the root apex. This is interpreted to indicate that the physiological basis for nodule initiation is distinct from that for initiation of lateral roots. The role of a single tetraploid cell in nodule initiation is refuted, as is the existence of incipient meristematic foci in the root. It is suggested that the tetraploid cells in nodule meristems arise from pre-existing endoreduplicated cells, or by the induction of endoreduplication in diploid cortical cells by Rhizobium.     

In vitro studies on the anatomy and morphology of bud regeneration in melon cotyledons

Summary The anatomy and morphology of bud regeneration were investigated in melon (Cucumis melo L.) cv. Galia, which regenerates in vitro only by direct organogenesis from the cotyledon explant. Explants were cut from the cotyledon proximal to the apex from 3-d-old in vitro seedlings. After 3 d on Murashige and Skoog medium with N⁶-benzyladenine, cell division can be observed in the epidermal layer on the adaxial side in the center of the explant, near the most proximal (wounded) cut edge. Over the next week, the area of the meristem increases laterally. Additional cell layers are added to the meristematic area by cell division in the epidermis. In places the epidermis remains active in cell division. Alongside those active areas there are zones where the epidermis has become inactive, although the subepidermal layers continue to divide. In transverse section, the explant now has small protuberances on the adaxial surface. After 10 d on cytokinin-containing medium, the first signs of development are visible on the adaxial surface adjacent to the proximal cut edge. The protuberances observed after 10 d are neither primordia nor buds, although some meristematic bulges are observed. The first regenerated shoot buds are observed histologically after 15 d, by which time the surface has many protuberances and some small leaves. The first shoot is found by histology after 22 d. By this time the surface is covered with protrusions and leaves, mostly without accompanying buds. The leaves may be produced from the protrusions initially visible after 10 d.     

Optimization of in vitro multiple shoot clump induction and plantlet regeneration of Kentucky bluegrass (Poa pratensis)

An efficient protocol for Kentucky bluegrass (Poa pratensis L.) in vitro culture was established using shoot apices of seedlings as explants. The optimal procedure of this protocol for majority of the genotypes was that meristematic cell clumps and small calluses were firstly induced from the bases of explants on initial culture medium supplemented with 0.9 μM 2,4-d and 8.9 μM 6-BA for 20 d, then were separated and transferred to shoot clumps induction medium containing 8.9 μM 6-BA for the formation of multiple shoot clumps. The percentage of multiple shoot clumps and numbers of shoots per clump were deeply related with the combinations of different plant growth regulators, duration of initial culture, the intensity of illumination and genotypes. Histological observation of the induced explants revealed that the meristematic cell clumps were produced from repeated division of the cortical cells and original meristematic primodium cells of explants, and the multiple shoots were formed via organogenesis pathway in the meristematic cell regions of cultures on shoot clumps induction medium. In this study, plantlets were efficiently regenerated on large scale from seven cultivars of Kentucky bluegrass. Hence the meristematic cell clumps and small calluses in this protocol could be considered good targets for genetic transformation of Kentucky bluegrass.     

Histological study of callus formation and root regeneration from mung bean (Vigna radiata W.)

We have established a reproducible culture system for callus formation and root development from juvenile stem segments of mung bean(Vigna radiata). In particular, we have studied the influence of plant growth regulators. Induction of calli from young stem explants was very effective on MS inorganic salts supplemented with 0.5 mg/L 2,4-D and 1.0 mg/L kinetin. In regenerating adventitious roots from callus tissues, we found that a combination of 0.75 mg/L NAA, 1.5 mg/L kinetin, and MS salts resulted in 20% efficiency. Histological examination showed that callus tissues originated from out-growths of the cambium rings through de-novo meristematic activity. Those rings were localized outside the vascular cambium. Adventitious roots that developed from root primordia originated from the center of the Callus masses. These primordia produced tracheid-like cells, which then became meristemoid cells for the cambium. Newly formed adventitious roots had the typical tetrarche actinostele type.     

Effect of cytokinins on plastid development and photosynthetic polypeptides during organogenesis ofPinus ponderosa Dougl. cotyledons culturedin vitro

InPinus ponderosa Dougl., application of the cytokinins, benzyladenine and 2-isopentenyl adenine, to excised cotyledons, promoted thein vitro formation of meristematic centers which led to bud and shoot production. Meristematic cells showed plastids with poorly developed thylakoid membranes and rudimentary grana, whereas cells in non-meristematic tissues and in growth regulator free medium, had chloroplasts with well developed inner membranes, and more thylakoid membranes and grana than plastids of meristematic cells. Chlorophyll and six polypeptides associated with photosynthesis were present in lower concentrations in cytokinin-treated cotyledons than in those cultured in growth regulator free medium. Both benzyladenine and 2-isopentenyl adenine are effective in inhibiting the accumulation of at least two photosynthetic polypeptides in the first 24 h in culture. The ability of cotyledons to respond in this way to cytokinins is lost after three days in culture in growth regulator free medium prior to treatment with cytokinin.     

金桔组织培养中愈伤组织和芽形成的组织细胞学观察

用金桔茎段为外植体,培养在附加1.0毫克/升BA和0.l毫克/升IBA的MS培养基上,诱导愈伤组织和芽形成。观察了愈伤组织和芽形成过程中的组织细胞学变化。培养一周后,在茎组织切口两端开始膨大,细胞增大和开始分裂。培养两周后,开始形成瘤状愈伤组织。在愈伤组织中有形成层状分生组织、维管组织结节和分生细胞团。培养四周后,表层的分生细胞团分化形成大量芽原基,同时愈伤组织深层也出现分生细胞团。带节茎段可从切口两端的愈伤组织分化形成芽,亦可从叶腋的潜伏芽直接形成芽。     

Histological study of in vitro development of adventitious buds on leaf explants of oriental beech (Fagus orientalis lipsky)

Summary Adventitious shoots were induced on the proximal portion of leaves excised from Fagus orientalis shoot cultures derived from a 2-mo.-old or a 4-yr-old seedling. Up to 90% of the explants formed between 13 and 19 buds after culture on Woody Plant Medium containing 2.9 μM indole-3-acetic acid and 4.5 μM thidiazuron. Adventitious buds developed mostly on petiole stub callus, but also on the midvein. The histological events leading to shoot organogenesis were examined. Some shoots developed directly from subepidermis or epidermis, but most originated indirectly from cell file proliferation produced by periclinally dividing cells subadjacent to the epidermis. Some cells in the outermost layers of these files became meristematic and divided extensively, resulting in the formation of meristemoids after 16 d of culture. Dedifferentiation into meristematic cells, which exhibited a large, prominent nucleus, densely-stained cytoplasm, and a high nucleus-to-cell area ratio, was generally associated with anticlinal divisions in cells previously originated by periclinal division. Subepidermal cell proliferation occurred mainly in the abaxial surface of the explant, which initially formed a diffuse cambium and later evolved to a phellogenic cambium. Some meristemoids were also differentiated by lenticel phellogen. Organized cell divisions in meristemoids gave rise to bud primordia that emerged from the explant surface and differentiated a protoderm. The progressive structural differentiation of the apical meristem, leaf primordia, and procambial strands led, after about 28 d of culture, to shoots with vascular connections with treachery elements previously differentiated in adjacent tissues.     

Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.)

Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x10⁴ protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.     

栝楼不定根尖分化不定芽过程中的细胞组织学研究

将栝楼（Trichosanthes kirilowii)长约0.5-1cm不定根尖（连同原外植体茎段或根段一起,或不连）培养在MS附加6－BA5mg/L的培养基上光照培养15d,可在根尖分化出大量不定芽。不定根尖培养过程中每隔2－3d取材,用FAA固定液固定1次,通过石蜡切片观察,将栝楼的不定根尖端分化不定芽的细胞组织学变化分为4个时期。1．启动期（0-3d),根尖分生组织细胞、中柱鞘细胞起动分裂。2“根茎转变区”原形成层形成期（4－6d0。起动细胞分裂后形成2－3层体积小、核大、质浓、近似扁平形的细胞层,组成“杯形”的“根茎转变区”原形成层。3．“根茎转变区”形成期（7－10d)。原形成层不同部位加速分裂使根尖膨大成半球形、球形或梭形,并在膨大区进行维管组织的转变。4．芽分化形成期（11－15d)。原形成层在不同部位向外形成“突起”即分生细胞团,每个“突起”发育为1个芽原基。作者还讨论了栝楼与其它植物根芽产生的异同。     

Histology of somatic embryogenesis in cultured immature embryos of maize (Zea mays L.)

Summary The developmental histology of somatic embryo (=embryoid) formation in cultured immature embryos of hybrid maize cultivars (Zea mays L.) is described. Embryos cultured on media containing 2% sucrose formed distinct globular embryoids. These embryoids arose either directly by divisions confined to the epidermal and the subepidermal cells at the coleorhizal end of the scutellum or from a soft and friable embryogenic callus produced by them. On media containing 6% sucrose divisions were initiated in the cells adjacent to the procambium of the cultured embryos. Subsequently, zones of meristematic cells also were observed in the region of the node and in the basal portion of the scutellum. Mature, well organized somatic embryos as well as a compact nodular type of embryogenic callus were produced as a result of localized meristematic activity along the tip of the scutellum toward the coleorhiza. Some embryos formed only the compact type of callus, and shoot primordia were organized later in the surface layers of this callus.Abbreviations CH casein hydrolysate - MS Murashige and Skoog's nutrient medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Changes in macromolecular movement accompany organogenesis in thin cell layers of <Emphasis Type="Italic">Torenia fournieri</Emphasis>

A range of fluorescently labelled probes of increasing molecular weight was used to monitor diffusion via the symplast in regenerating thin cell layer (TCL) explants of Torenia fournieri. An increase in intercellular movement of these molecules was associated with the earliest stages of vegetative shoot regeneration, with the movement of a 10 kDa dextran (FD 10000) observed between epidermal cells prior to the appearance of the first cell divisions. A low frequency of dextran movement in thin cell layers maintained under non-regenerating conditions was also observed, indicating a possible wound induced increase in intercellular movement. Dextran movement between epidermal cells reached a peak by day 4 of culture and then declined as cell division centres (CDCs) formed, became meristematic regions and finally emerged as adventitious shoots. Within CDCs, testing with small fluorescent probes (CF: carboxyfluorescein, mw 376 Da and F(Glu)₃: fluorescein-triglutamic acid, mw 799 Da) revealed a mosaic of cell isolation and regions of maintained symplastic linkage. Within shoots, surface cells of the presumptive apical meristem permitted the intercellular movement of 10 kDa dextrans but epidermal cells of the surrounding leaf primordia did not permit dextran movement. In some cases, intercellular movement of CF was maintained within leaf primordia. Symplastic movement of labelled dextrans during regeneration in Torenia thin cell layers represents a significant increase in the basal size exclusion limit (SEL) of this tissue and reveals the potential for intercellular trafficking of developmentally related endogenous macromolecules.     

Shoot preformation in clones of Fraxinus pennsylvanica in relation to site and year of bud formation

Summary Shoot preformation was investigated in buds of four clones of Fraxinus pennsylvanica var. subintegerrima (Vahl) Fern. at two sites in Manitoba in the second (1988) and third (1989) growing seasons after grafting. More preformed primordia were produced in terminal buds in 1989 compared to 1988 at each site. Both terminal and lateral buds at Morden contained significantly more primordia than those at Winnipeg. The numbers of preformed primordia were significantly different among clones. Clone 3 produced the most and clone 1 the fewest primordia in terminal buds. Despite quantitative variation, the pattern was similar among clones for terminal buds at each site and in each year. A similar pattern was evident for lateral buds at the Winnipeg site in 1989 but at Morden, clones 4 and 1 had the largest number of preformed primordia. Data from 1989 revealed that numbers of primordia were correlated with bud dimensions, parent shoot length, diameter and number of leaves, and location of the bud on the parent. Shoot dry weight was also related to these variables and revealed a non-linear increase in dry weight with shoot length. Multiple regression, with parent shoot length and location of buds along the parent axis as independent variables provided a reliable indicator of preformation in the crown. Although there is a genotypic component to preformation, variation between sites, years and crown locations suggests plasticity in bud development.     

Histology of Shoot Bud Ontogeny from Seedling Root Segments of Brassica napus L.

Root explants of Brassica napus cultured in vitro form adventitiousshoots. The root buds originated at the base of the newly initiatedlateral root. Cells in association with the differentiatingphloem of the developing lateral roots were the sites for rootbud formation. A nodular mass of cytoplasmic cells developedby day 7 at the base of the lateral root. This group of cellscontinued to divide an enlarge. The cells in the peripheralregion of the nodular cell mass differentiated further intoa meristematic zone. The meristematic cells grew towards theperiphery of the cortex by crushing the outer layer of corticalcells. Further development of the meristematic layer resultedin the formation of shoot primordia with organized shoot apicalmeristems and leaf primordia.Copyright 1993, 1999 Academic Press Brassica napus, canola, cultured root segments, root buds     

大岩桐花萼切块离体培养直接再生花芽的形态观察(简报)

前文发现在基本培养基上添加赤霉素和细胞分裂素能促进离体培养的大岩桐花蕾直接再生花芽[1],后又发现细胞分裂素能协同赤霉素促进离体培养大岩桐花萼切块高频率直接再生花芽[2,3],所得结果提示,这可能是一个研究赤霉素和细胞分裂素在花分化发育中作用的良好实验系统。为了完善这一实验系统,明确离体培养的大岩桐花萼切块培养物直接再生花芽的起源以及花分化的形态进程是十分必要的。为此我们对离体培养大岩桐花萼切块的形态变化在体视显微镜下进行了系统观察,并进行了石蜡切片及电镜扫描观察。进行大岩桐(Sinningia speciosa Hiern)花萼切…