Tissue Culture of Maize I. Callus Induction and Growth

Callus induction and subculture was successful with mature embryos and stem sections of seedlings of Zea mays L. on Linsmaier and Skoog's medium modified to contain 4 mg/I of 2,4-D and 1 g/I of casamino acids. — 2,4-D was superior to NAA and IAA for both callus induction and growth. Callus subcultured on NAA formed abundant roots on agar-solidified media and numerous root-like primordia in liquid cultures. — Kinetin had no effect on callus induction in the presence of 2,4-D and neither kinetin nor gibberellic acid stimulated callus growth during subculture. — Callus grew equally well on the medium of Linsmaier and Skoog, that of Schenk and Hildebrandt, and the B-5 medium of Gamborg and Eveleigh containing 2% sucrose, 4 mg/I of 2,4-D and 1 g/I of casamino acids. — The callus grew more rapidly at 25°C than at 30°C or 35°C. Little difference was noted at any temperature in callus growth in alternating light (16 h) and dark (8 h) or continuous dark. — Sucrose was superior to glucose and maltose in both liquid and agar-solidified cultures. Lactose and galactose failed to support callus growth.     

Callus induction and plant regeneration from shoot portions of mature embryos of high tannin sorghums

Callus and plant regenertion were induced from shoot portions of mature embryos (dry seeds) of five high tannin Sorghum bicolor (L.) Moench cultivars. The explants were cultured on Murashige and Skoog medium with altered concentrations of 5 salts, supplemented with 150 mg/L L-asparagine, 5mg/L 2,4-Dichlorophenoxyacetic acid and 0.05mg/L kinetin. Calli which were yellow and globular were formed with 70–90% frequencies. The subculture medium which gave best results was MS with 2mg/L 2,4-Dichlorophenoxyacetic acid and 0.5mg/L kinetin. Plants were regenerated on MS medium supplemented with 150mg/L L-asparagine and 0.2mg/L kinetin with regeneration frequencies of 11–48%.Abbreviations 2,4-D dichlorophenoxyacetic acid     

Rooting and the Metabolism of Nicotine in Tobacco Callus Cultures

The usefulness of exogenous nicotine as a factor in the induction of morphogenesis in a tobacco tissue culture medium has been demonstrated. Nicotiana rustica callus cell cultures were grown on a modified Murashige and Skoog medium with 2 mg/l indoleacetic acid (IAA) and 0.2 mg/l kinetin (MMS). Root morphogenesis was induced in roller tube callus cell cultures and solid callus cell cultures grown on MMS without kinetin supplemented with 10–100 mg/l nicotine. Optimal nicotine concentration for root induction was 50 mg/l. Other tests using varying combinations of IAA, kinetin and nicotine produced no obvious morphogenesis, although some changes in the amount of callus growth and endogenous protein concentration did correlate with nicotine concentration relative to the presence of IAA and/or kinetin. In liquid MMS medium, ¹⁴C-nicotine was primarily incorporated into the protein fraction of cultured cells while primarily incorporated into the cell wall and/or cell membrane fraction of cells cultured on MMS without kinetin in the medium. In MMS without IAA and MMS without both IAA and kinetin, there was incorporation, but to a lesser extent in both the protein and the cell wall and/or cell membrane fractions.     

Regeneration of garlic plants (allium sativum L., CV. “Chonan”) via cell culture in liquid medium

Summary Aiming at the genetic improvement of garlic cultivars, a cell suspension protocol was established which includes the induction of friable callus, establishment of cells in liquid medium, plating, regeneration, and bulb formation. Calluses of various textures from compact to friable and from green to yellowish were obtained by culturing explants excised from inner leaves of garlic bulbs on Marashig-Shoog (MS) medium with 2,4 dichlorophenoxy acetic acid (2,4-D), (1.1 mg/liter [5.0 μM]), picloram (1.2 mg/liter [5.0 μM]), and kinetin (2.1 mg/liter [10 μM]). Friable callus occurred on MS-A contained 2,4-D alone (1.0 mg/liter [4.52 μM]) and this callus was used to develop cell suspension cultures, which were maintained in liquid MS-B medium with a 2,4-D/benzyl adenine (BA) (0.5 mg/liter [2.25 μM]: 0.5 mg/liter [2.22 μM]) ratio. High plating efficiency was obtained on MS-C medium with different naphthalene acetic acid/BA combinations. Regeneration occurred after transfer of the caulogenic mass to MS-C medium containing 10 mg/liter (74.02 μM) and 20 mg/liter (148.04 μM) adenine for 60 days, followed by transfer to adenine-free medium. Plantlets transplanted to soil showed normal phenology. Shoots grown on modified MS medium supplemented with indolylbutryic acid (3.0 mg/liter [14.7 μM]) stimulated bulb formation by 30 days in culture.     

SHOOT AND TREE PRODUCTION FROM ASPEN TISSUE CULTURES

Trees were produced from firm white callus tissue of triploid quaking aspen (Populus tremuloides Michx.), initiated on a modified Wolter and Skoog defined medium and subcultured monthly for two years. When subcultured to medium without auxin, kinetin or supplements, but containing 0.15 mg/liter BAP (6-benzylaminopurine), multiple stunted shoots appeared on most inocula. However, at 0.05 mg/liter BAP, only a few vigorous shoots per piece were initiated, but seven rooted on the callus: two in the dark with BAP and five in 200 ft-c of light with 0.04 mg/liter 2,4-D. After proliferation of the roots on the medium surface, four shoots elongated and were planted in semi-sterilized soil, then were given 3100 ft-c of light for rapid growth into trees. Both light sources were on for 16 hr/day. Two trees were also grown from stunted shoots excised from the callus and rooted in soil.     

Plant regeneration fromBrassicanigra (L.) Koch stem protoplasts

Summary Protoplasts ofBrassica nigra (L.) Koch were isolated from stem peels of bolting racemes and cultured in 1.5 ml of VN1 liquid medium. The protoplasts in the liquid medium were plated on top of half strength MS medium supplemented with 400 mg/liter glutamine, 15 mg/liter glutathione, 50 mg/literl-serine, 0.25 mg/liter 6-benzylaminopurine, 0.5 mg/liter 2,4-dichlorophenoxyacetic acid, 1.5% sucrose, and 5% mannitol, pH, 5.7, solidified with 0.3% agarose. Ten percent of calli obtained from the protoplasts developed into plantlets within 4 wk after transfer onto 2N regeneration medium which contains MS salts plus 200 mg/liter casein hydrolysate, 0.625 mg/liter 6-benzylaminopurine, 0.625 mg/liter kinetin, 0.625 mg/liter 6-(γ,γ-dimethylallylamino)-purine, 0.625 mg/liter zeatin, 0.5 mg/liter 1-naphthaleneacetic acid, 1.5% sucrose, and 0.4% agarose. THis is the first report of plant regeneration fromB. nigra protoplasts.     

Callus Induction from Explants of Crocus sativus

Saffron calli were induced from ovary explants on Murashige-Skoog (MS) medium supplemented with beyzyladenine (BA) and naphthalene acetic acid (NAA) as growth factors. MS medium with 5 mg l^?1 BA and 10 mg l^?1 NAA was selected for calli induction and undifferentiated calli growth, while MS medium with 1 mg l^?1 BA and 1 mg l^?1 NAA was the most appropriate for stigma differentiation. On this medium, stigma-like structures measuring 0.5–1.5 cm were obtained. Initially they were colourless, but yellow pigmentation, due to the presence of crocin, progressively increased with calli growth. Extracts of stigma-like structures were analysed by HPLC and the presence of saffron secondary metabolites was demonstrated. In addition, calli also showed yellow pigmentation.     

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; )     

Shikonin derivative formation on the stem of cultured shoots in Lithospermum erythrorhizon

Lithospermum erythrorhizon , which are capable of producing red pigments, have been established. The red pigments were formed on the stems of L. erythrorhizon shoots cultured both on solid and in liquid media without phytohormones at 25 °C in the dark. Thin-layer chromatography, high-performance liquid chromatography and ¹ H nuclear magnetic resonance analyses revealed that the red pigments which accumulated on the cultured shoots were shikonin derivatives. The effects of various basal media and phytohormones (indole-3-acetic acid, indole-3-butyric acid and kinetin) on the growth and the formation of shikonin derivatives were investigated. When the shoots were cultured on Murashige and Skoog solid medium, the addition of kinetin remarkably enhanced shikonin derivative accumulation in the shoots. However, these effects of kinetin were not observed in the liquid culture when cultured in Gamborg B5 medium. The maximum content of shikonin derivatives (2.3% as dry weight, ca. 1.5 mg/100 ml flask) was observed in the shoots cultured in phytohormone-free B5 liquid medium for 5 weeks. Received: 1 February 2000 / Revision received: 23 March 2000 / Accepted: 28 March 2000     

Effects of N-1-naphthylphthalamic acid on the growth and bud formation of tobacco callus grown in vitro

The effect of N-1 -naphthylphthalamic acid (NPA), indole-3-aceticacid (IAA) and kinetin on callus growth and bud formation wasstudied mainly by a tobacco callus culture method. Callus producedfrom Nicotiana tabacum var. Wisconsin 38 was used as the testplant material. Callus growth on nutrient agar containing 2mg/liter of IAA was promoted by NPA added at a concentrationof 0.5 mg/liter with 0.4 mg/liter of kinetin or by NPA addedat 5 mg/liter in the absence of kinetin. At a high concentrationof 50 mg/liter, however, NPA inhibited growth on the mediumcontaining 2 mg/liter IAA and no kinetin. Kinetin reduced thisNPA inhibition. In the presence of 0.4 mg/liter kinetin and2 mg/liter IAA, when the concentration of NPA was 50 mg/liter,buds were initiated after calluses were grown on the test mediumfor 7 weeks in dim light, but no buds formed when NPA was omittedfrom the above medium. The control of callus growth and bud initiation is based onthe active ratio of auxin (IAA) to cytokinin (kinetin) in themedium and NPA added to the medium can promote or inhibit callusgrowth and induce bud formation. Therefore, it is proposed thatNPA can itself reduce auxin activity or enhance cytokinin activityand hence change the active ratio of the two regulators. NPAmay enhance the activity of cytokinin (here supplied as kinetin)but cannot substitute for it. ¹Present address: Department of Biology, Wisconsin State University,Oshkosh, Wisconsin 54901, U. S. A. (Received March 10, 1969; )     

Nutritional and Hormonal Requirements for the Growth of Vigna sinensis Callus in vitro

Nutritional and hormonal requirements for in vitro growth of callus tissue of Vigna sinensis Endl. were studied. Callus was formed on hypocotyl and root sections, when they were cultured on Linsmaier and Skoog's basal medium solidified with 10 g/1 agar and supplemented with only 0.5 mg/12,4-D or 2 mg/1 IAA. Further addition of 0.2 mg/1 kinetin and 1 g/l yeast extract resulted in more active callus formation. For unlimited vigorous growth of subcultured callus which was originally isolated from root sections, yeast extract was indispensable besides 2,4-D and kinetin. Such growth-promoting activity was observed also in malt extract and Ebios (dried cell powder of brewery yeast). Of known compounds tested, nicotinic acid, nicotinamide., methyl nicotinate and NAD were promotive to the growth of the callus, although much less effective than yeast extract. Other pyridine derivatives, vitamins and amino acids tested were ineffective or slightly effective. Sucrose was the most suitable carbon source. Fructose, glucose and maltose also supported the growth. Kinetin stimulated cell proliferation of the callus and cell differentiation to tracheary element.     

In vitro shoot tip multiplication of cowpea Vigna unguiculata (L.) walp

Summary Shoot multiplication was induced in cowpea, cv. Georgia-21, from shoot tip explants. Shoot tips, 5 mm long, were isolated from in vitro-grown seedlings and cultured on MS medium containing N⁶-benzyladenine (BA) at 1, 2.5, or 5 mg/liter (4.4, 11.1, or 22.2 μM) or 6-furfurylaminopurine (kinetin) at 1, 2.5, or 5 mg/liter (4.6, 11.6, or 23.2 μM) combined with 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.3 μM) or naphthaleneacetic acid (NAA) at 0.01, 0.1, or 0.5 mg/liter (0.05, 0.5, or 2.7 μM). Cultures were maintained at a 12-h photoperiod (40 μmol·m⁻²·s⁻¹) and 23 ± 2° C. Treatments with BA induced greater shoot proliferation than those with kinetin. The highest number of shoots was produced on 5 mg (22.2 μM) BA per liter in combination with NAA or 2,4-D at 0.01 mg/liter (0.05 μM). Callus proliferated from the basal ends of shoot pieces in all treatments. The cultures also formed roots in the presence of kinetin, but not on BA-containing medium. To produce whole plants, the shoots were separated and rooted on 0.1 mg (0.5 μM) NAA per liter. Resulting plants grew normally under greenhouse conditions. Shoot tips provide an excellent explant source for cowpea micropropagation and can be used for callus induction.     

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 ROOTING OF LIQUID-GROWN ASPEN CALLUS

Liquid-grown callus was used to study the nutritional requirements for rooting of a triploid form of normally diploid quaking aspen (Populus tremuloides Michx.). In modified Wolter's liquid medium, tan rough-surfaced spheres of callus grew rapidly when supplied with a high concentration of 2,4-D (0.5 mg/liter), but light-yellow uniformly smooth and firm spheres grew more slowly with a low level of 2,4-D (0.04 mg/liter) plus kinetin. When tissue was grown uncut in liquid for 1, 2, or 3 months, then subcultured to a low-2,4-D agar medium, rooting increased primarily with the age of the source tissue rather than the initial explant size. The surface of the youngest tissue source was almost smooth, but free columnar proliferations extended out from the surface for two or three cells in 2-month-old tissue and for three to six cells in the 3-month-old tissue. The relationship of increased rooting with increased surface cell-proliferation of older tissue was not determined anatomically.     

朱砂根愈伤组织诱导及其岩白菜素含量的测定

取朱砂根(Ardisia crenota Sims．)幼苗不同部位作外植体,研究不同激素组合对愈伤组织诱导的影响,利用薄层层析(TLC)和高效液相色谱(HPLC)检测愈伤组织中岩白菜素含量。实验结果表明朱砂根幼苗的不同部位(胚根、下胚轴、茎及叶)均能诱导出愈伤组织,最高诱导率都在45％以上,以MS基本培养基添加2,4—D0．5mg L^-1和KT0．01mg L^-1时愈伤组织的诱导和生长最好。这些愈伤组织均有合成岩白菜素的能力,其中以胚根诱导的愈伤组织中岩白菜素含量最高,为干重的0．076％,相当于原植物根中含量的1／9。高效液相色谱定量测定法精密度高,专一性好,能准确检测朱砂根愈伤组织中岩白菜素的含量。     

Effects of Chemical and Physical Conditions on Growth of Camptotheca acuminata Cell Cultures

Callus was induced from Camptotheca acuminata, which produces an antitumor alkaloid, carnptothecin. Using the Murashige and Skoogs’ medium as the basal, cultural conditions were examined for C. acuminata suspension cultures. As a result, a medium, containing 0.1 mg/liter 2,4-D, 3 mg/liter kinetin and 0.05 mg/liter GA₃, was established as a medium that gave the best cell growth in suspension cultures. In addition, conditioning of medium and addition of 0.115 mm l-Trp and l-Phe to medium promoted remarkably growth of cell suspensions.     

Production of pigments byMonascus purpureus using sugar-cane bagasse in roller bottle cultures

Solid-state fermentation, using sugar-cane bagasse, and submerged fermentation, using a semi-synthetic medium, were performed for pigment production byMonascus purpureus in both stationary and rotary conditions. Rotary cultures gave higher yields of crude red and yellow pigments than stationary cultures whereas twice the amount was synthesized at an earlier time (day 8) in liquid medium (1,285U yellow pigment/bottle, 1,728U red pigment/bottle). Supplementing the liquid medium with 0.6% (v/v) corn oil doubled the extracellular pigment yield but halved fungal growth.     

High efficiency plant regeneration by somatic embryogenesis from callus of mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor)

Summary Tissue culture methods were developed for reproducible induction and maintenance of embryogenic (E) callus established from developmentally mature embryo explants of bread wheat (Triticum aestivum) and grain sorghum (Sorghum bicolor). Embryogenic callus was obtained by culturing seeds and mature embryos of wheat on Linsmaier and Skoog’s (LS) medium containing 5 or 2 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D), respectively, and for sorghum mature embryos on LS medium containing 2 mg/1 2,4-D plus 0.5 mg/liter kinetin. Plant regeneration from E callus was achieved for several months and quantified on a fresh-weight basis of E callus. Phenotypically normal plants were regenerated from E callus cultured on LS medium supplemented with 0.1 mg/liter IAA plus 0.5 mg/liter benzyladenine (BA) for wheat and 1.0 mg/liter IAA plus 0.5 mg/1BA for sorghum. Wheat research was funded by the United States Agency for International Development, Washington, DC, cooperative agreement DNA-4137-A-00-4-53-00. Sorghum research was supported by the Gas Research Institute, Chicago, IL, contract 5084-260-0973. Expert technical asistance was provided by Nitschka S. ter Kuile, Barbara J. Ashton, Laurie Osborne, Erin Scott, and Kathleen M. Petersen.     

Accumulation of Glutamine by Suspension Cultures of Symphytum officinale

A callus was induced from the veins of a leaf of Symphytum officinale, comfrey, on a medium containing the inorganic elements reported by Murashige and Skoog with addition of 3% sucrose, 0.5 mg/liter 2,4-D and 0.3~3.0 mg/liter kinetin.

Suspension cultures of this cell line obtained from the callus were shown to accumulate a large amount of L-glutamine intracellularly, The effect of growth hormones and nutrients on accumulation of the amino acid has been examined in suspension cultures. The most suitable concentrations of 2,4-D and kinetin for glutamine accumulation were 0.3 mg/liter each. The presence of potassium nitrate as a nitrogen source was beneficial for growth and ammonium nitrate stimulated the accumulation of glutamine. High levels of these nitrogen sources in the medium were required for obtaining a high level of glutamine. The concentration of glutamine accumulated reached to approximately 20% of dry cell weight when S. officinale was incubated in the medium containing 0.495 % of ammonium nitrate and 0.570% of potassium nitrate which corresponded to three times higher levels than those in a Murashige and Skoog’s medium.

Most of the amino acid was found intracellularly but a small amount was excreted into the medium in the later stages of the incubation. Addition of a cationic surfactant, cetyltrimethylammonium bromide, to the cultures caused to increase the amount of the amino acid in the culture filtrate.

The contents of free amino acids in leaves of S. officinale were compared with those in the callus. The level of glutamine in the callus was 260 times higher than that in the intact plant.     

NUTRITIONAL REQUIREMENTS OF FRAXINUS CALLUS CULTURES

A satisfactory synthetic medium has been developed for continuous growth of Fraxinus pennsylvanica Marsh. callus cultures. The medium contains modified Reinert and White (1956) inorganic nutrient solution with 5 mg/liter Fe as NaFe-EDTA and supplemented with myoinositol 10 mg/liter, pyridoxine HCl 0.1 mg/liter, 2,4-dichlorophenoxyacetic acid 0.04 mg/liter, kinetin 1 mg/liter, sucrose 20 g/liter, and agar 10 g/liter. myo-Inositol, pyridoxine and an auxin are essential. α-Naphthaleneacetic acid is an effective alternate auxin. Kinetin and to some extent gibberellic acid improve the yields. Thiamine has no effect.