Effect of kinetin on auxin uptake and distribution in normal and tumor-prone Nicotiana shoots

Stem segments of non-tumorous Nicotiana glauca and N. langsdorffiiplants and of their tumor-producing amphidiploid F₁ hybrid weretreated with 6-furfurylaminopurine (kinetin) prior to transporttests with applied labeled indoleacetic acid (IAA-2-¹⁴C). Kinetin-treatmentsincreased the uptake of IAA in non-tumorous shoots; the IAAuptake by N. langsdorffii segments was increased up to 3-fold.The auxin uptake in stem-segments of the tumor-forming hybrid,however, could not be increased significantly by kinetin. Theeven distribution of IAA-¹⁴C in segments of normal and tumorproneNicotiana shoots is stimulated by kinetin. Data are discussedin conjunction with previous results on auxin transport andtumorformation in Nicotiana. (Received August 8, 1972; )     

Cytokinin effects on growth of quiescent tobacco pith cells

Excised pith tissue from Nicotiana glauca or the tumor-prone hybrid N. glauca × N. langsdorffii has no growth requirement for exogenous cytokinins. Addition of kinetin to cultures of these lines results in growth inhibition at a kinetin concentration 1000-fold lower than the optimal level for kinetin-requiring lines. Cytological comparison of the kinetin-inhibited 2N hybrid and glauca tissues with pith from the kinetin-requiring N. tabacum var. Wisconsin 38 suggests that the nature of the cytokinin action is similar in both situations and that the primary function of cytokinin, when it stimulates growth, may be to curtail cell expansion, thereby facilitating a balance of cell expansion and division requisite for maximal growth.     

Somatic hybrids of plants by fusion of protoplasts

Summary Fusions of protoplasts from Nicotiana langsdorffii and Nicotiana glauca were induced using polyethylene glycol. Parasexual hybrid colonies were selected for their ability to grow without growth substances. Hybrid plants, regenerated after grafting, were all tumorous and exhibited morphological and chromosome number variations. Out of 48 colonies selected in vitro only 6 regenerated flowering plants. Two of these plants had 42 chromosomes and were morphologically identical to the sexual amphidiploid Nicotiana glaucaxlangsdorffii.     

Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of indole-3-acetic acid (IAA) in the enzyme extracts of Nicotiana glauca, Nicotiana langsdorffii, their F1 hybrid, their amphidiploid hybrid, and the nontumorous mutant of the hybrid was investigated. Tryptamine, a possible precursor of IAA biosynthesis in Nicotiana tabacum, was not found in the callus tissue of N. glauca, N. langsdorffii, and their F1 hybrid.

In petiole slices, the synthesis of IAA progressively increased during 5 hours of incubation in [¹⁴C]tryptophan. The rate of synthesis was about equal in the hybrid and N. langsdorffii but lower in N. glauca on either a cell or fresh weight basis. It was also found that tryptophan was about 25 times more efficient than tryptamine in promoting synthesis of IAA in petiole slices.

It was found that indoleacetaldehyde oxidase, indoleacetaldehyde reductase, and tryptophan aminotransferase activities were present in all of the species examined; however, tryptophan decarboxylase activity was not found. The tryptophan aminotransferase activity in N. glauca, N. langsdorffii, and the nontumorous mutant required α-ketoglutaric acid and pyridoxal 5-phosphate whereas the addition of pyridoxal 5-phosphate seemed not to increase the enzyme activity in tumor plants.

The tryptophan aminotransferase in the amphidiploid hybrid was partially purified by acetone precipitation. The enzyme activity had a temperature optimum at 49 C and a pH optimum at 8.9. It is suggested that there is an indolepyruvic acid pathway in the synthesis of IAA in the Nicotiana species examined.

     

Bound Form Indole-3-acetic Acid Synthesis in Tumorous and Nontumorous Species of Nicotiana

The synthesis of H₂O-soluble and NaOH-hydrolyzable bound forms of indole-3-acetic acid (IAA) in petiole slices of Nicotiana glauca, Nicotiana langsdorffii, and their tumorous and nontumorous hybrids in the presence of exogenous ¹⁴C-IAA was investigated. The synthesis of conjugates progressively increased during 6 hours of incubation in ¹⁴C-IAA. The results showed that the rate of synthesis of IAA conjugates was higher in tumorous hybrids supplied exogenous IAA than in the parental species similarly supplied, and the rate of synthesis was higher in amphidiploid tumor plants than in a nontumorous mutant. It was also found that after 10 to 12 hours of incubation, 45% of the IAA taken up by F1 hybrids was in conjugated form whereas only 10 to 25% of the IAA taken up by a nontumorous mutant, N. langsdorffii, or N. glauca was conjugated. An F1 hybrid and an amphidiploid hybrid were found equally efficient in conjugating exogenously supplied IAA. It is postulated on the basis of these and other findings that IAA conjugates play an important role in tumorigenesis in Nicotiana.     

Differential alcohol dehydrogenase and malate dehydrogenase isozyme expression in long-term callus tissue cultures ofCereus peruvianus (Cactaceae)

Alcohol dehydrogenase (ADH) and mitochondrial malate dehydrogenase (mMDH) isozymes were tested as markers to study the effect of a high kinetin concentration on isozyme phenotypes and on the development ofCereus peruvianus callus tissue culture. Three-year-old callus tissues were used as samples. Callus tissue samples grown on 4.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and on 4.0 and 8.0 mg/LN-(2-furanylmethyl)-1H-purine-6 amine (kinetin) were cut and transferred to fresh medium containing 4.0 mg/L 2,4-D and 4.0, 8.0, 16.0, and 32 mg/L kinetin combinations. The pattern of changes observed in the ADH and mMDH isozymes as well as the growth of callus tissues was independent of the concentrations tested. The various ADH and mMDH isozymes seem to be products of differential association of subunits of the twoAdh and twomMdh genes. Both genes are active throughout callus tissue development; however, gene expression changed with various callus culture conditions. This study addresses how long-term callus culture conditions affect constitutive and differential gene expression of theAdh andmMdh genes inC. peruvianus.     

Physiological Comparisons of Pith Callus With Crown-Gall and Genetic Tumors of Nicotiana glauca, N. langsdorffii, and N. glauca-langsdorffii Grown in Vitro. II. Nutritional Physiology

Explants of genetic tumors, tumors initiated by Agrobacterium tumefaciens strains B-6 and T-37, and excised pith plugs from Nicotiana glauca, N. langsdorffii, and N. glauca-langsdorffii were cultured on Murashige and Skoog medium. All cultures, pith callus and tumors with the exception of N. langsdorffii pith grew on this medium. Addition of glutamine to the medium resulted in highly organoid growth in N. langsdorffii pith. In order to have material comparable to other pith cultures, N. langsdorffii was initiated on 2,4-dichlorophenoxyacetic acid medium, after which it grows on complete medium as amorphous pith callus. Except for the initiation of N. langsdorffii (and N. glauca) pith, the 2,4-dichlorophenoxyacetic acid medium, caused bleaching in cultures of T-37 induced tumors and death of B-6 induced tumors. Tumor cultures, except for the seedling tumor, grew well on a minimal medium lacking kinetin, indoleacetic acid, vitamins, glycine, and inositol. Glycine was necessary only in the growth of N. langsdorffii pith callus. A tissue culture model is presented which permits comparison of the various tissue types.     

Caffeic acid-O-methyltransferase in a suspension of cell aggregates of tobacco

Aggregates of tobacco cells in suspension in 2,4-D (10^?6 M) and kinetin (10^?5 M) cultures were fractionated by size, then their O-methyltransferase (OMT) activities were assayed. Only the kinetin culture showed high OMT activity, which was higher in the larger than the smaller aggregates at all stages of cell growth. The contents of phenolic acids were also greater in the larger cell aggregates in the kinetin culture. However, when the kinetin cultured cells were transferred to a medium containing 10^?6 M of 2,4-D, the relationships between the cell size of the aggregates and OMT, lignin and the phenolic acids disappeared. The importance of kinetin and cell association for OMT and the subsequent lignification of the cells is discussed.     

Somatic hybrid plants of Nicotiana glauca and Nicotiana tabacum obtained by protoplast fusion

Somatic hybrid plants were produced by fusion of protoplasts from cell cultures of the Nicotiana tabacum L. sulfur mutant Su/Su and from leaf mesophyll of Nicotiana glauca Graham. After fusion the N. glauca protoplasts failed to survive under the selected culture condition. From the hybrid cells light green shoots were produced. The hybrid plants exhibited intermediate characters between parental species with respect to leaf morphology, trichome density, floral structure and flower color. The chromosome number of 25 hybrid plants was 2n = 72 and both N. glauca and N. tabacum chromosomes were identified in the hybrids. Results of isoenzyme analysis showed bands of both parents and a specific (hybrid) band for aspartate amino-transferase. Small subunit fraction-1-protein of somatic hybrids also consisted of the sum of N. glauca and N. tabacum bands. Leaf spot formation associated with the Su locus of N. tabacum was observed in somatic hybrids.     

Effects of hornonal balance during leaf-disc infection of Nicotiana plumbaginifolia with Agrobacterium tumefaciens nopaline strain C58

Abstract

The technique of leaf-disc transformation has been used in order to study the influence of different hormonal balances on the process of transformation of Nicotiana plumbaginifolia tissues with Agrobacterium tumefaciens nopaline-strain C58.

C58 Agrobacterium strain incites withish, compact undifferentiated calli on Nicotiana tabacum, whilst allows the recover of shoots in N. plumbaginifolia. Shooting can be modulated through the hormonal balance of the culture medium mostly during the tissue culture phase (phase 2) subsequent to the period of infection (phase 1). Complete absence of hormones in phase 1 and 2 does not result in any callus formation or shooting on the edges of the infected leaf-discs, thus indicating that in this case the transformed cells need hormones at least in initial phase of development. In both the phases, addition of 2,4-D, NAA and kinetin resulted necessary in the stimulation of callus and shooting, namely in phase 2, and kinetin played a determinant role. Direct shooting also leads to an higher frequency of NOS⁺ regenerated shoots.     

Plant regeneration via somatic embryogenesis in mature embryo-derived callus culture of Sinocalamus latiflora (Munro) McClure

Somatic embryogenesis and subsequent formation of plantlets was achieved from callus cultures derived from mature zygotic embryos of Sinocalamus latiflora (Munro) McClure (Bamboo). Embryogenic callus was initiated on Murashige and Skoog's medium (MS) supplemented with 6 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 3 mg/l kinetin, 250 mg/l polyvinylpyrrolidon and 5% sucrose. Prolonged culture of the embryogenic callus on the same medium resulted in embryoid formation. The embryoids developed further to yield whole plantlets when transferred to a medium containing lower concentrations of 2,4-D (3 mg/l) and kinetin (2 mg/l).     

Caryological behavior during the first phases of dedifferentiation and habituation inNicotiana bigelovii

Summary Hypocotylar tissues ofNicotiana bigelovii var.quadrivalvis seedlings were induced to dedifferentiate and to habituate after a short treatment with 2,4-D or kinetin.Investigation by cytophotometry and chromosome counts on the nuclear cytology of callus induction and development in minimal medium showed, in addition to diploid cells, an high incidence of aneuploid nuclei. Amitotic phenomena have frequently observed during the culture period. The sequence of nuclear events were not influenced by the hormonal composition of the medium.The origin of these aneuploid cells from nuclear fragmentation processes is hypothesized.The results are discussed in comparison with the cytological status of other habituated and tumorous tissues in plant and animal systems.     

ORIGIN OF ADVENTITIOUS SHOOTS REGENERATED FROM CULTURED TOBACCO LEAF TISSUE

Leaf discs from Nicotiana tabacum L., N. glauca Grahm., and a series of interspecific periclinal chimeras were cultured on Murashige and Skoog (MS) medium containing either 2.5 mg/1 6-benzylaminopurine(BA) or 3.0 mg/16-furfurylaminopurine (kinetin). Most shoots regenerated from chimeral leaf discs were non-chimeral but 51 of 658 shoots were chimeral. The histogenic composition of plants regenerated from leaf discs of periclinal chimeras indicated that any cell layer in a leaf can be the ultimate source of shoots, and that shoots can be multicellular in origin. Certain periclinal arrangements were recovered more frequently and their chimeral nature was more stable during subsequent shoot growth. While N. tabacum leaf discs regenerated shoots on MS medium supplemented with either BA or with kinetin, N. glauca leaf discs did not form shoots on the medium containing kinetin. However, chimeras which possessed cells of both species arose on medium containing BA or kinetin, indicating that the morphogenetically competent (i.e., able to produce shoots in culture), N. tabacum cells either interacted with N. glauca cells leading to their competence or incorporated non-competent N. glauca cells into nascent shoot apical meristems.     

Significance of caffeic acid-O-methyltransferase in lignification of cultured tobacco cells

Caffeic acid-O-methyltransferase, activity was assayed in the callus and suspension cultured cells of tobacco. Lignification of cells was observed only in a kinetin (10^?5 M) culture and not in an auxin (10^?6 M 2,4-D or 10^?5 M IBA) culture. Enzyme activity in the kinetin cultured cells was much higher than in the stock culture and the rise in enzyme activity coincided with the onset of lignification.     

Callus Induction in Small Flowered Willow Herb (<Emphasis Type="Italic">Epilobium parviflorum</Emphasis> Schreb)

In order to determine the most suitable in vitro tissue culture and plant regeneration conditions for the small flowered willow herb (Epilobium parviflorum Schreb), various explants were cultured on semi-solid MS media containing factorial combinations of plant growth regulators. Callus induction from hypocotyl, cotyledon, petiole and leaf explants was achieved on media containing 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (KIN). All other growth regulator combinations [□-naphtaleneacetic acid (NAA) ± benzylaminopurine (BAP), NAA ± thidiazuron (TDZ), indol acetic acid (IAA) ± Zeatin (ZEA)] tested failed to respond. The best results with cotyledon- and petiole- derived callus were obtained from MS medium supplemented with 1.0 mg l^?1 2,4-D + 0.1 mg l^?1 KIN and 2.0 mg l^?1 2,4-D + 0.2 mg l^?1 KIN. It was observed that B5 basal medium was more effective than MS basal medium for producing seedling and the most effective seed sterilizing solution was 25 % (v/v) sodium hypochlorite (NaOCl). No plant regeneration was observed in either callus induction or during the subculturing stage. This is the first report on in vitro tissue culture study within the genus Epilobium.     

Correlation between the presence of membrane-bound auxin binding and root regeneration in cultured tobacco cells

When cell-suspension cultures and callus tissue from Nicotiana tabacum are grown on medium containing -naphthaleneacetic acid (NAA) and kinetin, three classes of auxin-binding proteins can be detected. When the herbicide 2,4-dichlorophenoxyacetic acid is used instead of both NAA and kinetin, one of these sites, which is membranebound, disappears. After retransferring cells to medium containing NAA and kinetin, this membrane-bound site reappears after four to eight weeks. This reappearance is correlated with the ability of the cells to regenerate roots.Abbreviations IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Effect of exogenous plant growth regulators on in vitro seed growth,embryo development,and haploid production in a cross between H. vulgare (L.) and H. bulbosum (L.)

Exogenous plant growth regulators are known to increase the efficiency of interspecific and intergeneric crosses. In vitro floret culture provides a defined system for assessing the importance of various plant growth regulators on the determinants of haploid production efficiency (seed set, embryos per seeds, and plants per embryos) in Hordeum vulgare × Hordeum bulbosum crosses. The individual and combined effects of three plant growth regulators (2,4-D, GA₃ and kinetin) on in vitro seed growth, embryo development and haploid production efficiency were tested in floret culture of the cross H. vulgare, cultivar Klages × H. bulbosum. All treatments, except kinetin alone, produced larger seeds and more embryos/100 seeds than the control (no plant growth regulator). 2,4-D alone was superior to GA₃ alone in haploid production efficiency (70.6 vs. 51.5) as measured by the number of plants regenerated/100 florets pollinated. Although kinetin +2,4-D+GA₃ produced the largest seeds and embryos, no advantage over 2,4-D alone was observed in haploid production efficiency. 2,4-D alone or kinetin +2,4-D are recommended for the purpose of barley haploid production in floret culture using the bulbosum method.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

Induction of indoleacetic Acid synthetases in tobacco pith explants

Formation of indoleacetic acid synthetases in tobacco pith explants was determined by following the growth of tissue cultures under conditions of indole-3-acetic acid (IAA) deprivation and by measuring the enzymatic conversion of tryptophan to IAA in the cultures. The pith explants obtained from the parent plant (Nicotiana glauca) and from basal regions of the tumor-prone hybrid (N. glauca × N. langsdorffii) both show a requirement for exogenous IAA for growth initiation in culture. The parent pith requires the constant presence of added IAA for continued growth, but hybrid pith, after initial treatment with IAA, will grow without further additions. IAA synthetases are detected in the cell homogenates of hybrid pith explants cultured with either continuous or initial IAA addition. These observations indicate that IAA may induce its own production. In contrast, IAA synthetases are not found in the parent pith under comparable culture conditions. Besides IAA, nonhormonal compounds such as indole and tryptophan are also capable of stimulating growth of hybrid pith, possibly through the induction of IAA synthetases needed for IAA formation. Indole and tryptophan are, however, inactive in growth promotion of the parent pith. These results suggest that the genomic expression of IAA synthetase formation is more stringently controlled in N. glauca than in the tumorprone hybrid.     

High frequency somatic embryogenesis and synthetic seed production of the endangered species Swertia chirayita

An efficient protocol for plant regeneration through somatic embryogenesis was established from in vivo leaf explants of Swertia chirayita, a critically endangered medicinal herb. The highest frequency (76%) of embryogenic callus was induced on Murashige & Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L kinetin (Kn) from in vivo leaf explants. Globular somatic embryos were induced and further matured from such embryogenic calli by subsequent culture on the same medium. The highest number of somatic embryos (48.83 ± 4.6) was recovered from embryogenic calli derived from leaf explants after 6 weeks of culture. Synthetic seeds were produced by encapsulating of torpedo stage embryos in sodium alginate (4% W/V) gel, dropped into 100 mM calcium chloride (CaCl₂ · 2H₂O) solution. The synthetic seeds were germinated on MS medium. The highest frequency of synthetic seed germination (84%) was observed on MS medium supplemented with 1.0 mg/L BA and 0.5 mg/L NAA. Regenerants were successfully acclimatized under ex vitro condition. This is the first report on synthetic seed production of S. chirayita. Application of these protocols would be helpful in reducing stress in natural habitat, and in long-term storage of elite genotypes through synthetic seed production.     

The establishment of cell suspension culture of sabah snake grass (<Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Burm.F.) Lindau)

Clinacanthus nutans (Burm.F.) Lindau is an herbaceous plant that has long been used for traditional medicinal purposes in Asia. It has recently gained popularity as an alternative treatment for cancer. The aim of this study was to establish cell suspension cultures of C. nutans and to identify targeted bioactive compounds in the cultures. Young leaf explants were cultured on Murashige and Skoog medium supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin to identify a suitable medium for callus induction and proliferation. Proliferated, friable calluses were cultured in different combinations of plant growth regulators (2,4-D, naphthaleneacetic acid [NAA], picloram, kinetin, and 6-benzylaminopurine) in liquid medium to establish cell suspension cultures. Three cell lines of suspension culture, callus, and intact plant parts were subjected to ethyl acetate extraction followed by thin layer chromatography for identification of selected bioactive compounds. Medium supplemented with 0.25 mg L^?1 2,4-D and 0.75 mg L^?1 kinetin was found to be optimal for callus induction, whereas supplementation with 0.50 mg L^?1 2,4-D was efficient for callus proliferation. Liquid medium supplemented with 0.25 mg L^?1 2,4-D and 0.50 mg L^?1 NAA produced the highest growth index (2.52). Quercetin, catechin, and luteolin were present together in the callus and cell suspension cultures of C. nutans, but all three compounds were detected separately in young leaves, mature leaves, and stems. This study is the first to report the establishment of cell suspension culture of C. nutans with both cell and callus cultures producing quercetin, catechin, and luteolin.