Suppression of the Tumorous State in Crown Gall Teratomas of Tobacco A Clonal Analysis

Two types of experimental approaches designed to test the possible involvement of chimeras in the persistent but readily reversible suppression of the tumorous state and in a recovery from that state in crown gall teratomas of tobacco were investigated. These studies showed that the tumorous state was suppressed in specialized cells present in the different tissues derived from each of the three layers of the apical meristem of teratoma-derived shoots of clonal origin. Although differences existed in the exogenous requirements needed for the reestablishment of the tumorous state in the different specialized cell types present in the different tissues, all again acquired a capacity for autonomous growth when appropriately stimulated. No evidence was found for the existence of periclinal or sectoral chimeras in teratoma-derived shoots of a single cell origin. Chimerism was, however, found to exist in one noncloned teratoma-derived shoot. Analysis of more than 500 clones derived from leaf mesophyll cells of cloned teratoma shoots showed that the vast majority of them (> 99%) were tumorous, while all of the 144 seed-grown plants from these shoots had completely recovered from the tumorous state. The results of these studies suggest that (a) a teratoma shoot does not depend for its development on the presence of significant numbers, if any, of cells that had recovered from the tumorous state; (b) the tumorous state may be suppressed in even the most highly specialized cells; (c) some special mechanism exists in the meiotic process for the elimination of all or most of the foreign DNA from transformed cells.     

在1/3海水培养基上筛选豆瓣菜耐盐变异体

The responses of stem segments of watercress ( Nasturtium offtcinale R. Br. ) to 6-BA, NAA and 2,4-D were studied. MS medium supplemented with 2.0 mg/L 6-BA, 0.2 mg/L 2,4-D was used for callus initiation and maintainance. MS medium supplemented with 4.0 mg/L 6-BA was suitable for plant regeneration and MS medium without plant hormone supplement was used for rooting and plant propagation. For screening of salt. tolerant calli, stem segments of watercress were plated onto callus initiation medium containing 1/3 natural seawater. Seventeen out of the 325 plated explants produced calli. The growth curves demonstrated that the growth rate of salt-tolerant calli on saline medium almost matched that of the control calli on normal medium. Some of the salt-tolerant calli were transferred to the normal regeneration medium or saline regeneration medium to induce plant regeneration. In the first case, buds and shoots were regenerated in the same way as those of control calli on normal regeneration medium. More than 1 000 regenerated shoots were obtained of which 83 regenerated shoots were cut and transferred to saline MS base medium. At first, all shoot growth was inhibited, but 40 days after the transfer, rapid-growing axillary shoots were observed on 16 of the original shoots but none on the control shoots on saline MS base medium. Moreover, green spots appeared on most calli 10 days after they were transferred to saline medium, however buds appeared only on 5 calli from the 30 transferred calli and at the end only 2 rapid-growing shoots were obtained from two calli. In total, 18 variant lines were obtained through propagation of the salt-tolerant shoots on saline MS base medium. RAPD analysis was performed in 10 of the 18 salt-tolerant variant lines and DNA variation was detected in all the tested variant lines.     

Plant tumor reversal associated with the loss of foreign DNA

Summary Transformation of plant tissues into crown gall tumors has been associated with the transfer of a portion of a tumor-inducing plasmid (Ti-plasmid) into plant DNA. Various laboratories have regenerated normal-appearing plants from a number of crown gall tumors. This study investigates the fate of the foreign DNA in a series of tissues derived from various parts of a plant regenerated from the tumor BT-37 by Braun and his coworkers. It was found that all the foreign DNA sequences were lost from tissues that had lost all their tumorous traits; whereas the plasmid DNA sequences were still present in tissues that appeared normal but still exhibited tumorous traits when returned to tissue culture media. From these studies it would appear that the presence of the Ti-plasmid sequences in the plant DNA is required for the maintenance of the transformed state. Presented in the Symposium on Gene Transfer, Differentiation and Neoplasia in Plant and Animal Cells at the 30th Annual Meeting of the Tissue Culture Association, Seattle, Washington, June 10–14, 1979. This symposium was supported in part by Grant CA 26748 from the National Cancer Institute, DHEW, and Grant RD-67 from the American Cancer Society.     

Multiplication of tobacco mosaic virus in tobacco callus tissues and in vitro selection for viral disease resistance

Tobacco mosaic virus-resistant tobacco was selected in vitro using callus tissues induced from axillary buds of systemically infected tobacco plants. Callus lines in which the virus was continuously multiplying were first isolated and redifferentiated into shoots. By the procedure, non-diseased, healthy shoots were successfully isolated from diseased shoots, which showed typical mosaic symptoms of the virus, and regenerated into intact plants.These regenerated plants showed resistance to virus inoculation, and selfed progeny of virus-resistant regenerants segregated the resistance and susceptibility according to the Mendelian system.     

烈香杜鹃的离体培养和高效植株再生

以烈香杜鹃嫩茎为外植体,应用均匀设计法筛选其最适合的嫩茎基部直接再生芽苗和生根培养基。结果表明,最适合烈香杜鹃嫩茎基部直接再生芽苗的诱导培养基为DR＋2．30mg·L-1 TDZ＋0．05mg·L-1 IAA＋1．80mg·L-1 KT,诱导率为99．6％;生根培养基为1／2DR＋0．07mg·L-1 IAA＋0．02mg·L-1NAA,生根率达99．7％以上。以再生植株茎节为材料进行快速繁殖,在35d的培养周期内,每段增殖倍数平均达5以上。     

The study of the conditions for the fertilizationin vitro in maize

Suitable conditions for the fertilizationin vitro in maize have been studied. The germinating capacity of pollen in synthetic media was low; it was confirmed that it might be stimulated by supplementing agar with egg yolk. Application of pollen onto styles overhanging from the culture medium of excised ovaries was examined. After 5 days the styles could be cut off the ovaries, for the pollen tubes had already penetrated the embryo sacs. However, better results were obtained when cultivating ovaries along with segments of the maize cob. Solid media were more suitable for the development of kernels. Some of them germinatedin situ and gave rise to normal plants. From nucellar meristems of young kernels a callus could be derived which, on further cultivation, became green and regenerated shoots and roots. The cells of meristems exhibited a varying number of chromosomes.     

Studies on the expression of marker genes in chickpea

Conditions were established for the optimum transient expression of beta-glucuronidase and neomycin phosphotransferase II genes introduced into zygotic embryos of chickpea (Cicer arietinum L. 6153 and CM72) by accelerated tungsten particles. Plasmid DNA at a concentration of 12 microgram per milligram of tungsten particles when accelerated with an inflow of helium gas at 60 kilogram per square centimeter through a distance of 24 centimeter in a chamber maintained at a negative pressure of 71.12 centimeter of mercury, resulted in optimal transient expression of the beta-glucuronidase gene in chickpea embryos. However, the expression of the marker genes was 20-40% higher under a cauliflower mosaic virus promoter in comparison to the Win6 and actin promoters. When Agrobacterium tumefaciens was used to transfer marker genes into zygotic embryos and the resultant plants were analysed for activity of the beta-glucuronidase and neomycin phosphotransferase II genes, both of these genes were expressed in tumorous tissues. When a disarmed strain of Agrobacterium was used, normal shoots were regenerated in which the lower parts showed expression of both genes at a frequency of 20%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enhanced Formation of Roots and Subsequent Promotion of Growth of Shoots on Cryopreserved Nodal Segments of Asparagus officinalis L

This study was designed to investigate the effects of cryopreservation on the survival, organogenesis, and growth of plants regenerated from nodal segments of asparagus (Asparagus officinalis L.) that had been cut from cultures in vitro. The addition of dimethylsulfoxide (Me2SO) to the freezing solution at 8-16% (v/v) with or without a sugar (glucose, sorbitol, or sucrose) was effective for successful cryopreservation by a slow prefreezing method. Frequencies of root formation (average, 59.3%) from cryopreserved and surviving nodal segments were significantly higher (P < 0.005) than those (average, 13.9%) from nodal segments that had only been treated with freezing solution supplemented with 12% (v/v) Me2SO and various sugars without freeze-thawing. The increased frequency of root formation from cryopreserved nodal segments appears to have been induced by the freeze-thaw step of the cryopreservation procedure. Numbers and lengths of shoots derived from cryopreserved nodal segments were initially lower but were subsequently higher, after 60-90 days of culture, than those of shoots derived from nodal segments without freeze-thawing. The promotion of growth of shoots from cryopreserved nodal segments seemed to have been due to the increased percentage of root formation. Histological observations revealed that only dome-shaped meristematic tissue and a few cells of cladophyll primordia survived in cryopreserved nodal segments that had been cultured for 5 days after thawing. Many mitochondria and well developed rER were observed in these cells. Disorganization and/or physiological changes might have occurred in the surviving tissues and/or cells of the cryopreserved nodal segments that were responsible for the subsequent increased formation of roots. Copyright 1998 Academic Press.     

Comparative study of different cytokinins in the induction of morphogenesis in lentil (Lens culinaris Medik)

Summary This study presents the first comparative analysis of the effect of four different cytokinins, applied to different lentil (Lens culinaris Medik.) explants in four different concentrations. with regard to the regeneration of shoots and roots of the pulse crop. The variables explant, phytohormone and concentration were all found to be highly significant. Mature seed explants showed the highest shoot regeneration over all the phytohormones and concentrations tested. Thidiazuron (TDZ) and benzyladenine (BA) showed a higher number of regenerated shoots than kinetin (KIN) and zeatin (ZEA); an increase from 1.25 μM to 10 μM of any cytokinin in general doubled the number of shoots regenerated. The average length of regenerated shoots was found to be inversely proportional to the number of shoots regenerated. TDZ and BA were found to inhibit root development more than KIN and ZEA. The highest root regeneration frequency was obtained from shoots regenerated on media containing 1.25 μM ZEA. The study concludes that in order to obtain whole plants it is best to regenerate shoots on media containing the cytokinins KIN or ZEA at low concentrations, in order to be able subsequently to regenerate roots.     

Tumorization-Redifferentiation System of Tobacco Genetic Tumor

Genetic tumor was easily and rapidly induced by cutting treatmentof tobacco (Nicotiana glauca ? N. langsdorffu) F₁ seedlingsin a simplified system in vitro. When this tumor was culturedunder weakened light intensity normal shoots developed frequently.After these shoots were maintained under appropriate light conditions(ca. 2–3 W/m²) they regenerated into whole plants withnormal appearance. The regenerated plants were identical tothose developed after germination with respect to their responseto cutting treatment. Using this system we are able to switchthe morphology between the tumorous state and the normal staterapidly and with ease. The significance and the implicationsof the availability of this system for studies of the mechanismof conversion between these two states at the molecular levelare discussed. (Received March 30, 1988; Accepted September 22, 1988)     

A new,endosperm-supported callus induction method for wheat (Triticum aestivum L.)

A new, endosperm-supported callus induetion method was developed using mesocotyls of mature wheat embryos. After seed germination under aseptic condition, most of the germ tissues were cut off and only a few mm of the mesocotyl tissue with the scutellum was used for callus induction. The seeds were placed furrow downwards in 2,4-D solution (6–8 mg l^-1). Proliferating callus tissues were already observed on the cut surface of the mesocotyls on the 2nd day after inoculation. On the MS nutrient medium, callus formation from the isolated scutella with attached mesocotyls was negligible even after 6 days. For shoot and root regeneration, the calli produced up to 10 days were removed from the seeds and transferred onto a hormone-free MS medium. As shown by histological methods, the plantlets regenerated via organogenesis.     

Somatic embryogenesis in Canary Island date palm

Shoot regeneration was obtained from leaves of in vitro cultures of wild pear genotypes. The highest regeneration rates, ranging from 40% to 64%, depending on the genotype, were obtained using leaves wounded by three cuts transversely to the mid-rib, a Quoirin and Lepoivre macro-salt composition, 250 mg l^-1 cefotaxime and maintaining the explants in darkness for the first 30 days (induction phase), then transferring them to an auxin-free medium in light (expression phase). A concentration of 8.8 μM BA induced the highest number of explants to produce adventitious shoots. TDZ was less effective than BA and induced hyperhydricity in regenerated shoots. The histological studies revealed that the regenerated shoots originated mainly from callus formed by epidermal and sub-epidermal cells and by cells of the vascular tissue. The regenerated shoots were micropropagated, rooted and transplanted to the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Introduction of a nitrogen-fixing cyanobacterium into tobacco shoot regenerates

Tobacco (Nicotiana tabacum L.) shoots associated with the nitrogen-fixing cyanobacterium Anabaena variabilis Kütz. (ATCC 29413) were regenerated in mixed cultures of tobacco callus and the cyanobacterium. The cyanobacteria were localized inside the tissues as well as on the surface of regenerated shoots, formed heterocysts, and were capable of acetylene reduction.     

Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants

 The green fluorescent protein (GFP) from Aequorea victoria has been introduced into three different citrus genotypes [Citrus aurantium L., C. aurantifolia (Christm.) Swing. and C. sinensis L. Osbeck×Poncirus trifoliata (L.) Raf.] which are considered recalcitrant to transformation, mainly due to low transformation frequencies and to the regeneration of escape shoots at high frequencies from the Agrobacterium-inoculated explants. High-level GFP expression was detected in transgenic cells, tissues and plants. Using GFP as a vital marker has allowed us to localize the sites of transgene expression in specific cells, always occurring in callus tissue formed from the cambium of the cut ends of explants. Whereas green fluorescent shoots regenerated in all cases from this callus, most escapes regenerated directly from explants with almost no callus formation. Thus, development of callus from cambium is a prerequisite for citrus transformation. Furthermore, in vivo monitoring of GFP expression permitted a rapid and easy discrimination of transgenic and escape shoots. The selection of transgenic shoots could be easily favored by eliminating the escapes and/or by performing shoot-tip grafting of the transgenic buds soon after their origin. GFP-expressing shoots have also been observed in citrus explants co-cultivated with Agrobacterium but cultured in a medium without the selective agent kanamycin. This opens the possibility to rescue the transgenic sectors and to regenerate transgenic plants without using selectable marker genes conferring antibiotic or herbicide resistance, which is currently a topic of much discussion for the commercialization of transgenic plants. Received: 28 October 1998 / Accepted: 28 November 1998     

A system in which anthocyanin synthesis is induced in regenerated torenia shoots

A system in which anthocyanin synthesis can be induced under defined conditions was established in regenerated torenia shoots. Leaf discs prepared from torenia plantlets grown under sterile conditions were placed on solidified half-strength MS medium containing 3% sucrose and 4.4×10^–6 M benzyladenine (BA) and cultured under 16 h light/8 h dark (standard light) conditions for 10 days, then in the dark for a further 10 days. The discs were transferred to medium containing 7% sucrose without BA and cultured under standard light conditions. Six days after transfer, anthocyanin synthesis started in the regenerated shoots, and thereafter, anthocyanin accumulation increased while chlorophyll content decreased. Experiments in which either the timing of illumination was altered or shoots were retransferred to medium containing 1.5% sucrose or other sugars as well as sucrose indicated that both osmotic stress and light are required to induce anthocyanin synthesis. Once anthocyanin synthesis was induced in the torenia shoots 6 days after transfer, the shoots were fated to the synthesis of anthocyanins and the degradation of chlorophylls, and could not revert to the developmental pathway of shoot regeneration. This system may provide a good model for the investigation of the mechanisms underlying the induction of anthocyanin synthesis.     

Cryptic floral determination: stem explants from vegetative tobacco plants have the capacity to regenerate floral shoots

The developmental fates of shoots regenerated in culture and in situ by stem tissues of Nicotiana tabacum cv. Wisconsin 38 from different positions along the main axes of plants at different ages have been characterized. It was expected that explants from vegetative plants would not have the capacity to produce floral shoots. Contrary to the expected result, a small percentage (about 0.2%) of the shoots formed from cultured stem explants taken from young, vegetative plants were floral, i.e., produced a small number of nodes and then a flower. A larger percentage (about 2%) of the shoots formed by explants from the same region of plants which had flowered were floral. The largest percentage (76%) of floral shoots arose from explants taken from the inflorescence. Internode cells which were stimulated to divide and undergo organogenesis in situ after decapitation of the plant also produced few-noded, floral shoots with apical internode tissues producing many such floral shoots and basal internode tissues producing few such floral shoots. These results indicate that the capacity to form a flower is a visible expression of a cryptic developmental state which is quantitatively but not qualitatively controlled in time and space.     

A radiotechnique suitable for the detection of octopine synthesis in Crown-gall tissues grown in vitro

An improved method for the detection of octopine in Crown-gall tissues and in shoots regenerated from these tissues has been devised using [¹⁴C]arginine as a precursor. By using bidimensional chromatography followed by autoradiography, very small amounts of octopine can be clearly detected in tumors grown in vitro. Using this method, octopine was detected in tumor tissues when usual techniques were not sensitive enough.     

The expression of tumour markers in intraspecific somatic hybrids of normal and crown gall cells from Nicotiana tabacum

Summary Following fusion of protoplasts from crown gall tumour calli, characterized by hormone independent growth, and protoplasts from normal tissues of a streptomycin-resistant mutant, SR1, we selected hormone independent streptomycin-resistant calli in Nicotiana tabacum. The tumour line, B6S3, lost the ability to form shoots. Some of the selected lines, similar to SR1, however, are morphogenic. Both calli and shoots contained the tumour specific enzyme lysopinedehydrogenase. The hybrid shoots are resistant to Agrobacterium infection and do not root. These tumorous properties are dominantly expressed in the somatic hybrids.     

Adventitious Shoot Regeneration in Carnation (Dianthus caryophyllus) from Axillary Bud Explants

Adventitious shoots were regenerated from axillary bud explantsof 15 carnation cultivars. The use of leaf and stem explantswas not successful, largely due to explant senescence in thepresence of benzyladenine, kinetin and, to a lesser extent,zeatin. For axillary bud explants, a suitable optimum adventitiousregeneration medium contained Murashige and Skoog basal mediumsolidified with Gelrite and supplemented with 15 µm benzyladenineand 0.5 µM a-napthaleneacetic acid. Adventitious primordiaarose from the cut basal end of bud explants erupting as individualshoots after 2–3 weeks incubation. The axillary bud sizeand the time between subcultures of source material influencedthe production of adventitious shoots. Transfer of regeneratedshoots onto a medium solidified with agar minimized visiblesigns of vitrification. Regenerated shoots could be easily rooted,transferred to glasshouse conditions and grown to flowering. Vitrification, tissue culture, cut flowers, Dianthus caryophyllus L., carnation, cytokinins, explant     

In vitro plantlet regeneration from seedling nodal explants of Acacia catechu

Multiple shoots were initiated after 20 days in stem nodes excised from in vitro grown seedlings of Acacia catechu, on Murashige and Skoog's medium adjuvanted with 1 to 100 microM of N6-benzyladenine (BA). Explants were subcultured on the same medium augmented with 1.5 g l(-1) of polyvinylpyrrolidone (PVP) after 30 days. In the second subculture, after 30 days, the explants were transferred to a medium lacking PVP, but containing 10 microM of BA, where nine or ten shoots differentiated per explant within next 30 days. If individual shoots along with some callus were subcultured on BA (10 microM), nearly 15 shoots per explant regenerated in 90 days. Thus, the average number of shoots obtained from each node was 142 after 180 days. Since a seedling develops four nodes after 20 days, theoretically an average of 568 shoots can be obtained from a single seed. If shoots were individually subcultured on 1/2-strength MS medium with 14.7 microM of indole-3-butyric acid (IBA), roots developed in 20 days. Addition of 40 mg l(-1) of glutamic acid to the rooting medium prevented leaf senescence. These plantlets thrived well in garden soil, sand and silica (1:1:1).