GROWTH REGULATOR EFFECTS ON BUD INITIATION IN CALLUS CULTURES OF MEDICAGO SATIVA

Influence of growth regulators on bud initiation in callus of alfalfa (Medicago sativa L.) was studied by varying levels and combinations in the first medium of a two-medium sequence used to obtain whole plants. Callus of tetraploid clone S-4 (cv. ‘Saranac‘) was initiated from immature ovaries on a modified Blaydes' basal medium containing all combinations of six concentrations (0–36 μM) of kinetin (K), six concentrations (0–44 μM) of naphthaleneacetic acid (NAA), and seven concentrations (0–36 μM) of 2,4-dichlorophenoxyacetic acid (2,4-D). After 28 days the callus was challenged to form buds by transfer to the modified Blaydes' medium containing 2.0 g/liter yeast extract and 0.57 mM inositol. No buds were produced in the absence of 2,4-D in the first medium, and the frequency of bud formation on the second medium was directly proportional to the 2,4-D concentration in the range 2.3–54 μM in the preceding medium. Buds were produced in the absence of kinetin in the first medium, but its presence in the range 2.3–36 μM markedly increased bud formation. NAA was not required for bud formation, and the budding frequency increased only slightly with increasing NAA concentration in the first medium. Budding of callus of two other alfalfa clones was also influenced by the 2,4-D concentration in the initial medium. There were several indications that many of the buds were initiated on the first medium and completed development on the second medium. These included the differential effect on budding of combinations of 2,4-D, NAA and kinetin in the callus initiation medium, the specific media sequence required, and the presence of embryoids on the callus which after transfer to the yeast extract-inositol medium produced buds.     

ANTHOCYANINS OF DIMORPHOTHECA (COMPOSITAE). I. IDENTITY OF PIGMENTS IN FLOWERS,STEMS, AND CALLUS CULTURES

Anthocyanins isolated from ray flowers, disk florets, stem, and callus cultures of Dimorphotheca sinuata were identified and found to be cyanidin-3-glucoside and dephinidin-3-glucoside. The anthocyanins of the callus cultures were of the same identity as those in the whole plant, demonstrating that these glucosides are produced in the same manner under autophytic and heterophytic conditions. The use of this callus culture in experimental studies of anthocyanin production is discussed.     

PRODUCTION OF ANOMALOUS STRUCTURES IN QUERCUS RUBRA L. CALLUS CULTURES

Internodal stem segments of 2–4-month-old Quercus rubra L. seedlings were cultured on Murashige and Skoog agar medium containing NAA and BA. Structures, termed organoids, were initiated from callus on medium containing 5 mg/1 NAA and 0.1 mg/1 BA. Organoids consisted of parenchymatous cells with a distinct epidermis and a continuous vascular system. Growth occurred principally by cell division throughout the parenchymatous tissue resulting in a variety of morphological shapes. Although no organized shoot meristems were observed, normal roots were produced; such roots were connected to the vascular system of the organoid.     

PRODUCTION OF YEAST VARIANTS BY AUXIN AND EFFECTS OF PLANT GROWTH REGULATORS ON THESE VARIANTS

Using diploid strains of Saccharomyces cerevisiae and S. ellipsoideus,the following facts were found:

1. Indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid and -naphthaleneaceticacid produced stable variants differing in the cell form andin the response to the actions of auxin to elongate cells, toinduce respiration- deficient mutation and to promote sporulation.
2. The auxins also produced stable variants differing in theabilityto form spores.
3. Acetic acid had no above-menthionedactions of auxin.
4. Spore-formation and cell elongation of someof auxin-inducedvariants were controlled by auxin.

Biological significance of the auxin-induced variation is discussedand the usefulness of some of these variants as experimentalmaterial for auxin physiology in general is pointed out. (Received November 1, 1966; )     

蓝光对绿豆下胚轴愈伤组织生长和呼吸的影响

与白光和黑暗条件相比,蓝光明显促进绿豆(Vigna radiata (Linn.)Wilczek)下胚轴愈伤组织的形成和生长。培养到18d,蓝光下生长的愈伤组织鲜重分别是白光和黑暗下的120％和191％。三种条件下愈伤组织的呼吸速率有明显的不同,培养5d,呼吸速率以蓝光处理的最高,白光处理的次之,黑暗处理的最低;培养14d,与上述相反,呼吸速率由高到低则依次是黑暗、白光和蓝光。应用呼吸抑制剂碘乙酸、丙二酸和正磷酸钠试验及对6-磷酸-葡萄糖脱氢酶活性测定的结果表明,培养5d,蓝光和白光处理时磷酸已糖支路(HMP)明显增加,被碘乙酸 丙二酸抑制后的呼吸占总呼吸的34％～36％,黑暗处理的仅占总呼吸9％～11％ 绿豆下胚轴愈伤组织中亦存在抗氰交替途径,黑暗处理时,此种途径的最大容量明显高于蓝光和白光处理的,培养14d,黑暗处理占总呼吸的31％左右.而蓝光和白光处理的则占20％以下。     

唐古特山莨菪毛状根中东茛菪碱产生的研究

以唐古特山莨菪的无菌苗叶为外植体,通过发根农杆菌诱导获得了唐古特山莨菪的毛状根培养系统,应用PCR方法鉴定了转化毛状根,并应用薄层扫描方法对唐古特山莨菪毛状根生物碱进行了测定,结果表明在液体培养毛状根的培养基中东莨菪碱含量可达10 mg/L,培养物中东莨菪碱的增加与莨菪碱的减少同步.     

胡萝卜光自养型愈伤组织的诱导培养及其光合特性

经诱导得到胡萝卜光自养型愈伤组织。以叶绿素为单位计算,获得的光自养型愈伤组织的光合活力达到甚至超过了整体植株叶片水平。同时测定愈伤组织光自养过程中光合特性的变化,结果表明其叶绿素含量逐渐上升、暗呼吸速率和Chl a/Chl b比值逐渐下降。并且用电子显微镜观察到愈伤组织中叶绿体结构逐渐发育的过程。     

ISOENZYME COMPLEMENTS OF DIANTHUS CALLUS CULTURES: INFLUENCE OF LIGHT AND TEMPERATURE

Callus cultures isolated from the stems of two clones of Dianthus were extracted for water-soluble proteins and the extracts were subsequently analyzed by acrylamide gel electrophoresis. Peroxidase, acid phosphatase, esterase, and 6-P-gluconate dehydrogenase (6-P-G DH) were studied under each of four combinations of light and dark, cold (0-5 C) and warm (25 C) environments. Marked differences in the isoenzyme patterns were observed under the four conditions with all the enzymes except 6-P-G DH. Acid phosphatase generally showed fewer isoenzymes than the usual stem complement, whereas the other enzymes showed the same or greater number of isoenzymes. Many of the isoenzymes from the callus tissue correlated well with those found in the stem; however, all but 6-P-G DH showed some different components. Of the four enzymes studied only peroxidase was recovered from the medium; these isoenzymes matched those found in the callus and in the stem. The isoenzymes in the medium were not influenced qualitatively by the growth conditions. The changes in the zymograms of the callus under the four environments were compared to the changes observed in the stem under stress environments.     

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURES

1. The longer the period of stock culture, the more remarkableis the growth inhibition by 8-azaguanine in callus.
2. Chloramphenicol,5-methyltryptophane and mitomycin C exert greaterinhibitionon growth in CCL than in CCS.
3. Bud formation is inhibited bysome concentrations of chloramphenicolwithout accompanyinginhibition of the growth.
4. Cell size and the contents of RNA,DNA, protein and lipid percell of CCL are greater than thoseof CCS, respectively. Thecontents per cell of RNA and lipidin "mitochondrial fraction"are higher in CCL than in CCS.
5. Incorporationof guanine-8-¹⁴C into RNA of CCS occurs rapidlyin the first12 hr and slows down thereafter, but that in CCL-RNAincreasessteadily for 16 hr. This difference in rate of theincorporationafter 12 hr between CCS and CCL is principallydue to the differencein rate of the incorporation into RNAof nuclear, mitochondrialand soluble fractions.

1. The rate of RNA breakdown in CCL wasnot so great as the rateof synthesis.
2. 8-azaguanine (10^–3and 10^–4M) inhibits incorporationof guanine-8.¹⁴C intoRNA of both CCS and CCL during 14 hr,but thereafter (up to25 hr) it inhibits the incorporation intoCCL-RNA alone leavingthat into CCS-RNA unaffected.

1. In CCL 510^–5M 8.azaguaninedoes not affect total radioactivityincorporated into bulk RNA,but inhibits incorporation intoRNA of "mitochondrial fraction".

(Received December 23, 1964; )     

ATTEMPTS TO OBTAIN BACTERIA-FREE PLANTS OF PSYCHOTRIA PUNCTATA (RUBIACEAE): GROWTH AND ROOT FORMATION IN CALLUS CULTURES

Attempts were made to obtain bacteria-free plants of Psychotria punctata from tissue cultures. Stem explants and callus derived from them were induced to form roots but failed to form buds on Linsmaier and Skoog medium and 96 chemical modifications of it, including most of those known to induce bud formation in other species. Roots formed with ample IAA (2 mg/liter or more) and a low kinetin concentration (0.25 or 0.50 mg/liter). Adenine inhibited root formation in these media, but tyrosine did not. Tyrosine did lower the percentage of calluses commencing growth. When enzyme-hydrolyzed lactalbumin (1.3 g/liter), kinetin (0.5 mg/liter) and IAA (5 mg/liter) were added to Linsmaier and Skoog medium modified by decreasing inorganic nitrogen and increasing inorganic phosphate, callus grew at the fastest rate observed (increasing threefold in fresh weight in three weeks) and formed numerous roots. This was adopted as the stock callus medium. Casein hydrolysates also stimulated growth but less so than lactalbumin hydrolysate. When lactalbumin hydrolysate or a casein hydrolysate lacking tryptophan was supplied, growth occurred without added auxin if sufficient cytokinin was added. Cytokinin was required at unusually high concentration and was tolerated at still higher concentration. Formation, elongation, and branching of roots persisted on a saturated solution of BA which inhibited callus growth about 70 % and delayed callus senescence. Light caused earlier callus senescence after growth had ceased but did not affect callus growth or root formation. Light-induced senescence was prevented by a high cytokinin concentration.     

THE IN VITRO SECRETION OF GROWTH REGULATORS BY ISOLATED CALLUS TISSUES

Tissues from a wide variety of plants were surface sterilized, isolated, and grown on different media. These isolated tissues were bioassayed for growth regulatory activity. The secretions from four of the 20 callus tissues inhibited the growth of Bacillus subtilis. An aseptic method for measuring the growth of Lemna was developed and used to detect inhibitory materials in medium which had supported the growth of five isolated callus tissues. In the seed (Lycopersicon esculentum) germination test five callus tissues had an inhibitory influence while two callus tissues showed a stimulatory effect. The study also included expressed juices and extracts of callus tissues which secreted regulatory materials. The expressed juice of five callus tissues contained materials which inhibited the growth of Lemna. Two expressed juices inhibited the growth of Bacillus subtilis. The water extract of two callus tissues inhibited the growth of Lemna. Fifty percent of the plants which have been reported to produce growth regulatory materials in nature also produced callus tissue which was capable of regulating growth of assay organisms.     

GROWTH AND THE PRODUCTION OF EXTRACELLULAR SUBSTANCES BY TWO STRAINS OF PHAEOCYSTIS POUCHETI1,2

Clones of Phaeocystis were isolated from winter surface waters off Woods Hole, Massachusetts, and from the tropical Atlantic near Surinam, South America. Northern strains survived only up to 14 C, while the tropical strain survived only as low as 17 C. Colony shapes of the northern and tropical clones differed somewhat, but the motile and non-motile single cells of both strains seemed identical in the light microscope. By current taxonomic criteria both strains belong to the species P. poucheti (Hariot) Lagerheim. When growing in the form of colonies, both strains excreted 16–64% of their photoassimilated carbon into the medium, mainly as carbohydrates of varying molecular weights. However, cultures predominantly in the form of single cells released only about 3% of their photoassimilated carbon. The qualitative composition of the carbohydrates released is similar for the 2 strains, consisting of some 8 sugars or sugar derivatives with glucose, mannose, and rhamnose as the dominant components. The production of acrylic acid was confirmed. We estimate that as much as 7 μg/liter of acrylic acid, and at least 0.3 mg/liter of polysaccharides can be liberated in a Phaeocystis bloom.