Effects of cytokinins on the auxin requirement and auxin content of tobacco calluses

High concentrations (0.1–1 mg/liter) of kinetin permittedcontinuous growth of auxin-requiring and cytokinin-nonrequiringtobacco calluses on a medium without auxin. This effect of kinetindid not seem to be due to perpetuating change in the tissuecharacter, because tissue was auxin-requiring when returnedto a kinetin free medium. Cytokinins, i.e. benzylaminopurineand geranylaminopurine, showed the same effect as kinetin inmaking auxin-requiring calluses grow without a supply of auxin. In auxin-requiring and cytokinin-nonrequiring calluses subculturedfor 3 years on a medium containing 1 mg/liter kinetin withoutauxin, at least 3 auxins were detected by bioassay; 2 in theacidic and 1 in the neutral fraction. One was identified asIAA by paper chromatography (bioassay), thin-layer chromatographyand gas chromatography. Reduced or no auxin activity was foundin calluses transferred to a medium without kinetin. Kinetinwas apparently required to maintain the endogenous auxin levelin callus tissues. Kinetin may act on the auxin requirement of callus via its effectson auxin metabolism. ¹ Part XVI in the series "Studies on Plant Tissue Cultures". (Received April 11, 1972; )     

Two effects of cytokinin on the auxin requirement of tobacco callus cultures

Cytokinin affects the requirement for auxin of a strain of tobacco callus (Nicotiana tabacum) which is cytokinin-autotrophic when grown on Murashige and Skoog medium with 11.4 mum of indole-3-acetic acid but requires cytokinin 6-(3-methyl-2-butenylamino)purine (i(6) Ade) when grown on the same medium with <3 mum indole-3-acetic acid. As the exogenous concentration of cytokinin (i(6) Ade) is increased, the concentration of indole-3-acetic acid required for growth is decreased. A second effect of cytokinin, observed sporadically in cultures with 2.5 mum or 5 mum i(6) Ade, is the transformation of some of the callus pieces to auxin-autotrophic growth. Strains, both callus-forming and bud-forming tissues, that arise in this manner are not permanently altered in their auxin requirement because subcultures on medium without cytokinin still require exogenous auxin.     

Evidence for a Mendelian factor controlling the cytokinin requirement of cultured tobacco cells

Cultured leaf tissues of Nicotiana tabacum L. cv. “Havana 425” normally require an exogenous source of cytokinin for rapid growth; stem-cortex tissues do not—ie, they exhibit the cytokinin-habituated phenotype. We found that plants regenerated from cloned cortex and leaf tissues from one particular plant differed in leaf-tissue phenotype: Leaf tissues derived from leaf cells exhibited the normal, nonhabituated phenotype, whereas leaf tissues derived from cortex cells were cytokinin-habituated. This difference in leaf phenotype was not found using leaf and cortex cells from six other donor plants. The inheritance of the habituated leaf trait was studied in tissues from cortex-derived plants and hybrids between these plants and normal plants. F₁ hybrids were intermediate between the parental types in degree of habituation. No differences were found between reciprocal hybrids. These results suggest that the habituated leaf trait is an incompletely dominant, nuclear trait. Both parental and intermediate phenotypes were recovered in the F₂ progeny. The frequency of habituated leaf progeny in the F₂ and backcross populations provide evidence that the trait is regulated at a single genetic locus.     

Effects of low temperature on induction and differentiation of wheat calluses

Young inflorescences and long-term culture calluses of wheat (Triticum aestivum L.) were used to investigate the effect of low temperature treatment on the potential of plant regeneration. The results indicated that the frequency of callus induction from immature inflorescences was decreased when treated for a long time at 5 °C. However, it was found that a 5 °C treatment significantly improved the regeneration frequency of calluses. The clear difference of peroxidases of wheat calluses was apparent after the cultures were treated at the low temperature. The isozyme band 8 became clearly faint, however, bands 6 and 9 became intense. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glucose-6-phosphate dehydrogenase in tobacco callus strains differing in their growth and their requirement for auxin and cytokinin

The cytokinin-autonomous strain (A_s) of tobacco callus differs from the original cytokinin-dependent strain (D) and from the cytokinin- and auxin-autonomous strain (A₄) by a significantly lower activity of glucose-6-phosphate dehydrogenase (G-6-PDH). Changes in the total G-6-PDH activity were associated with differences in the number of G-6-PDH isozymes. The A_s strain contained only one isozyme, four isozymes were found in D and A₄ strains.     

Stability of the cytokinin requirement in Paul's scarlet rose cells in culture

Summary Cells ofRosa sp. cv Paul's scarlet have been reported to require cytokinin for growth in suspension culture. This report was verified in the present study. However, a rose cell line that was maintained for 2 yr in suspension culture by routine subculturing developed the capacity to grow without exogenous cytokinin. The stability of the cytokinin requirement and the basis for this altered response to cytokinin was investigated. The parental cell line, which has been maintained independently on agar-solidified medium, was subcloned and the cytokinin dependence of the subclones was determined. The subclones were found to exhibit a continuous spectrum of responses, ranging from a high degree of cytokinin dependence for growth to rapid growth upon the initial transfer to cytokinin-deficient medium. The average growth constant (K=1n W/Wo; Wo=initial fresh weight,W=fresh weight after growth for the stated time interval) of 30 subclones grown on medium containing 0.5 μM zeatin was 3.1, with a range of 1.1 to 4.0. The average growth constant of the same subclones when grown on medium lacking a cytokinin was 1.5, with a range of 0 to 3.9. By comparison, the parental cell line exhibited growth constants of 3.5 in the presence of 0.5 μM zeatin and 1.6 in the absence of exogenous cytokinin. Although the growth of some of the subclones after transfer to cytokinin-deficient medium suggested that they were cytokinin autotrophs, this was not the case because none of them grew after a second transfer to medium lacking cytokinin. Culture in medium containing cytokinin conferred upon the cells the capacity for a limited amount of growth after subsequent transfer to medium lacking cytokinin. The extent of this cytokinin-induced growth potential varied from subclone to subclone. Efforts to determine the frequency with which cytokinin autotrophs appeared in a subclone that required cytokinin suggested that it is a rare event and that the cytokinin requirement is a fairly stable phenotypic characteristic of these cells. This research was supported by Grant PCM 7722398 from the National Science Foundation.     

忽地笑愈伤组织培养条件对加兰他敏合成的影响

研究了忽地笑次生代谢产物加兰他敏的合成以及各种理化因子对加兰他敏合成的影响。结果表明,蔗糖是忽地笑愈伤组织培养的最佳碳源,而且在90 g/L浓度培养下加兰他敏含量最高,达到0.068%;附加500 mg/L苯丙氨酸前体物也可提高加兰他敏产量。酪氨酸和水杨酸不利于加兰他敏的积累。此外,2,4-D和浓度高于0.5 mg/L的NAA、IBA抑制加兰他敏的合成,但高浓度的6-BA却对加兰他敏的合成起促进作用。     

温度变化对烟草光合作用光响应特征的影响

通过Li-6400光合测定系统控温,研究了大田条件下,烟草脚叶采烤期腰叶在17、20、25、30和35℃下的光合作用光响应特征。结果表明:随温度升高,净光合速率(Pn)、最大净光合速率(Pnmax)、初始斜率(α)和气孔导度(Gs)先上升后下降,在20℃下达最大值,35℃下净光合速率受强光的抑制作用明显;光补偿点(LCP)和暗呼吸速率(Rd)随温度升高而上升,但35℃下二者较30℃时有所下降;光饱和点(LSP)随温度升高而呈下降-上升-下降的变化,30℃和17℃时LSP较高;蒸腾速率(Tr)随温度上升而增强,水分利用效率(WUE)则随温度升高而下降,但20℃时水分利用效率在强光下明显较其他温度下的高。结果说明,20℃最适合烟草光合作用,此温度下气孔的水、气调节能力最强,温度高于30℃则对烟草光合作用不利。     

Effects of temperature on phyllody expression and cytokinin content in floral organs of rose flowers

Formation of leaf-like organs known as phylloids in Rosahybrida cv. Motrea flowers was promoted by exposure of plants toelevated temperatures. At a day/night temperature regime of26°C/21°C respectively theproportion of malformed flowers exhibiting phyllody was four times higher thanthat in flowers of plants grown at21°C/15°C. The number ofpetals in phyllody-expressing flowers was higher than that in normal flowers.The total content of endogenous cytokinins in young flower buds of plantsexposed to the lower temperature was six times higher than that at the highertemperatures. The effects of the reduced temperature were pronounced on all thegroups of cytokinins examined. However, the proportion of the various cytokiningroups remained similar at both temperature regimes. In contrast to thecytokinins in the flower buds, the content of all cytokinin types in youngleaves increased following exposure to the higher temperature and was reducedbythe lower temperatures. After 11 weeks at the lower temperature, about18% of the flowers remained malformed, whereas at the higher temperatureabout 20% of the flowers still remained normal. All thephyllody-exhibiting flowers were formed on vigorously grown basal shootscharacteristic to Rosa hybrida plants, whereas the normalflowers at the elevated temperatures were formed on lateral shoots which weremost distal to the plant base. However, irrespective of the season, thepresenceof normal and malformed flowers was observed on plants kept growing at standardconditions of 30°C/17°C inthegreenhouse. This phenomenon led us to examine the cytokinins in floral organsofnormal and malformed cv. Motrea flowers grown in the greenhouse as well as inflowers of a complete rose mutant known as a 'Green Rose(Rosa chinensis viridiflora). The highest content ofcytokinins was found in the pistils and stamens of normal 'Motreaflowers. On the other hand, the content of cytokinins in leaf-like style-tubesin the malformed flowers as well as in partially malformed ovaries at the baseof phylloids was significantly lower. A low content of cytokinins was alsopresent in petals of both normal and phyllody-exhibiting flowers and the lowestcontent has been found in the phylloids of the 'Green Rose. Apossibility of mutant deviations in metabolism of cytokinins in rose plants isdiscussed.     

Effects of senescence-induced alteration in cytokinin metabolism on source-sink relationships and ontogenic and stress-induced transitions in tobacco

Senescence and reserve mobilization are integral components of plant development, are basic strategles in stress mitigation, and regulated at least in part by cytokinin. In the present study the effect of altered cytokinin metabolism caused by senescence-specific autoregulated expression of the Agrobacterium tumefaciens IPT gene under control of the P_SAG12 promoter (P_SAG12-IPT) on seed germination and the response to a water-deficit stress was studied in tobacco (Nicotiana tabacum L.). Cytokinin levels, sugar content and composition of the leaf strata within the canopy of wild-type and P_SAG12-IPT plants confirmed the reported altered source–sink relations. No measurable difference in sugar and pigment content of discs harvested from apical and basal leaves was evident 72 h after incubation with (+)-ABA or in darkness, indicating that expression of the transgene was not restricted to senescing leaves. No difference in quantum efficiency, photosynthetic activity, accumulation of ABA, and stomatal conductance was apparent in apical, middle and basal leaves of either wild-type or P_SAG12-IPT plants after imposition of a mild water stress. However, compared to wild-type plants, P_SAG12-IPT plants were slower to adjust biomass allocation. A stress-induced increase in root:shoot ratio and specific leaf area (SLA) occurred more rapidly in wild-type than in P_SAG12-IPT plants reflecting delayed remobilization of leaf reserves to sink organs in the transformant. P_SAG12-IPT seeds germinated more slowly even though abscisic acid (ABA) content was 50% that of the wild-type seeds confirming cytokinin-induced alterations in reserve remobilization. Thus, senescence is integral to plant growth and development and an increased endogenous cytokinin content impacts source–sink relations to delay ontogenic transitions wherein senescence in a necessary process.     

A cytokinin mutant derived from cultured tobacco cells

Tissues cultured from the leaf lamina of wild-type Nicotiana tabacum L. cv. "Havana 425" plants require an exogenous source of cytokinin for rapid growth. In contrast, leaf tissues of plants heterozygous or homozygous for the partially dominant, monogenic habituated leaf (H1-1) trait, exhibit a cytokinin-autotrophic phenotype in culture. Here we show that the H1 trait can arise in culture. Cytokinin autotrophic variants were obtained by culturing wild-type tissues of leaf lamina successively on media containing reduced concentrations of the cytokinin, kinetin. Plants regenerated from clones of these variants exhibited the H1 phenotype, which segregated in breeding tests as expected for a dominant, monogenic trait. This trait, designated H1-2, is inherited at a different locus than the H1-1 trait described earlier. Our results show that cytokinin mutants can arise in cell culture and that at least two genes regulate the cytokinin requirement of cultured tobacco tissues.     

Effects of temperature on leaf hydraulic architecture of tobacco plants

Main Conclusion

Modifications in leaf anatomy of tobacco plants induced greater leaf water transport capacity, meeting greater transpirational demands and acclimating to warmer temperatures with a higher vapor pressure deficit. Temperature is one of the most important environmental factors affecting photosynthesis and growth of plants. However, it is not clear how it may alter leaf hydraulic architecture. We grew plants of tobacco (Nicotiana tabacum) ‘k326’ in separate glasshouse rooms set to different day/night temperature conditions: low (LT 24/18 °C), medium (MT 28/22 °C), or high (HT 32/26 °C). After 40 days of such treatment, their leaf anatomies, leaf hydraulics, photosynthetic rates, and instantaneous water-use efficiency (WUE_i) were measured. Compared with those under LT, plants exposed to HT or MT conditions had significantly higher values for minor vein density (MVD), stomatal density (SD), leaf area, leaf hydraulic conductance (K _leaf), and light-saturated photosynthetic rate (A _sat), but lower values for leaf water potential (ψ _l) and WUE_i. However, those parameters did not differ significantly between HT and MT conditions. Correlation analyses demonstrated that SD and K _leaf increased in parallel with MVD. Moreover, greater SD and K _leaf were partially associated with accelerated stomatal conductance. And then stomatal conductance was positively correlated with A _sat. Therefore, under well-watered, fertilized conditions, when relative humidity was optimal, changes in leaf anatomy seemed to facilitate the hydraulic acclimation to higher temperatures, meeting greater transpirational demands and contributing to the maintenance of great photosynthetic rates. Because transpiration rate increased more with temperature than photosynthetic rate, WUE_i reduced under warmer temperatures. Our results indicate that the modifications of leaf hydraulic architecture are important anatomical and physiological strategies for tobacco plants acclimating to warmer temperatures under a higher vapor pressure deficit.     

Induction of auxin-nonrequiring tobacco calluses and its reversal by treatments with auxins

Auxin-nonrequiring calluses were induced with high frequenciesby short treatments with auxins from auxin-requring ones. Auxin-requiringcalluses T22 and XD6S2, subcultured on a medium containing 1mg/liter IAA as plant growth regulator, required quite differenttreatment for induction from auxin-nonrequiring calluses; fromcalluses T22 and XD6S2, auxin-nonrequiring calluses were inducedby treatments with low concentrations of auxins (0.01–0.1mg/liter of IAA or NAA) and high concentrations of syntheticauxins (10–100 mg/liter of NAA or 1–10 mg/literof 2,4-D), respectively. No auxin-nonrequiring calluses wereobtained when they were transferred directly to the basal medium(hormone-free medium). The transformation of auxin-requiring into auxin-nonrequiringcalluses was fully reversible by treatment with 1 mg/liter ofIAA at an early stage of subculturing on the basal medium, butnot after prologed subculturing. ¹Part XXI in the series, ‘Studies on Plant Tissue Cultures’.Part XX, see Proceedings of the IVth International FermentationSymposium: Ferment. Technol. Today 697 (1972). (Received May 29, 1973; )     

Evaluation of the photosynthetic activity in transgenic tobacco plants with altered endogenous cytokinin content: lessons from cytokinin

Cytokinin is known to be involved in many processes related to plastid development and function but the exact role of cytokinin in photosynthesis remains elusive. To investigate more profoundly the effects of cytokinin in this process, the photosynthetic activity of transgenic Pssuipt and 35S:CKX1 tobacco (Nicotiana tabacum) plants with respectively elevated and reduced endogenous cytokinin content was evaluated. Pigment analysis indicated that elevated endogenous cytokinin content resulted in increased pigment content. Functional analysis of the photosynthetic apparatus by chlorophyll a fluorescence and in vitro electron transport measurements clearly showed that changing the endogenous cytokinin content affects the activity of the photosynthetic apparatus. Surprisingly, both an increase as well as a decrease in cytokinin content results in a better photosynthetic performance. Quenching analysis revealed that the initial responses of the photosynthetic apparatus on a dark-light transition are not affected by changed cytokinin content. However, it has an effect on the further kinetic behavior. Taken together, we suggest that cytokinins can induce structural changes in the different parts of the electron transport chain as also demonstrated by the in vitro electron transport measurements.     

The effects of a morphactin,chlorflurenol, on organ redifferentiation from tobacco calluses culturedin vitro

A certain morphactin, chlorflurenol (methyl-2-chloro-9-hydroxyfluorene-9-carboxylic acid) was found to be effective in inducing organ redifferentiation (shoot formation) from calluses derived from the pith parenchyma ofNicotiana tabacum ‘Wisconsin No. 38.’ The regenerative ability of chlorflurenol was strongly intensified in combination with kinetin. A combination of 1 mg/l of chlorflurenol and 2 mg/l of kinetin resulted in shoot formation in 70% of inoculated calluses. One reason for this high frequency of regeneration seems to be the promotion of tracheary elements, probably due to some synergism of chlorflurenol and kinetin. Chromosome numbers of the root-tip meristematic cells in regenerated plants varied widely over a range of 6 to 98. One regenerated plant showed normal chromosome constitution in the first metaphase of meiosis, and showed normal pollen and seed fertility; but the others remained unstable and had low pollen and seed fertility.