Isolated microspore culture of wheat (Triticum aestivum L.) in a defined media I. Effects of pretreatment,isolation methods,and hormones

Significant improvements were achieved in the production of haploid and doubled haploid plants from isolated microspore culture of wheat c.v. Chris on a defined media. Procedures found to be of benefit included: A 7-day pretreatment of anthers in 0.4M mannitol plus the macronutrients from FHG medium; the inclusion of 4.5 mg/liter abscisic acid in the pretreatment solution; the isolation of microspores from pretreated anthers by vortexing; and the use of phenylacetic acid (PAA) as the auxin source in MS medium. The best response was achieved with 4.0 mg/liter PAA in MS medium containing 90 g/liter maltose as the sugar source. Under these conditions, 68% of viable microspores underwent division, and an average of 93 embryos and 92 green plants were regenerated per 100 anthers used. The root-tip chromosome number and the fertility of 114 regenerating green plants revealed that 75% were completely fertile spontaneously doubled haploids.     

Optimization of somatic embryogenesis and embryo germination in soybean

Summary Somatic embryos of soybean [Glycine max (L.) Merr.] are induced on immature cotyledons explanted onto a medium containing moderately high levels of auxin. Germinability of embryos is related to morphologic normality, and both are reduced by excessive exposure to auxin during the induction process. Shoot meristem development was improved by reducing exposure of cotyledonary explants from 30 to 10 to 14 d on 10 mg/liter α-naphthaleneacetic acid (NAA). A 3-d exposure was sufficient to induce embryos, and embryo frequency was not significantly increased by exposures to NAA for more than 1 wk. Embryo frequency was enhanced, however, by transfer after 9 d to fresh medium containing 10 mg/liter NAA. Germination of morphologically normal embryos was achieved without growth regulators, after maturation for 1 mo. on hormone-free medium and desiccation for 1 wk in a sealed, dry container. This research was funded by Lubrizol Genetics, Inc., Madison, WI.     

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.     

Anthranilic acid and 3-indolyl-propan-1,2-diol as precursors of photorubin in Saccharomyces

Summary The formation of photorubin, the orange-red pigment produced by Saccharomyces, is due to a photochemical reaction between anthranilic acid and an indole derivative identified with 3-indolyl-propan-1,2-diol. These two compounds are excreted into the medium by strains partly or completely requiring pyridoxine which are grown in the presence of a suboptimal amount of this vitamin. If pyridoxine is added to the growth medium in amounts either sufficient or exceeding the needs of the cells (over 50 g per liter), or if the strain is auxo-autotrophic, a secretion of hypoxanthine occurs.     

Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose are investigated in an aerated stirred fermentor. Cellular growth first proceeds exponentially until exhaustion of ammonia in the fermentation medium. Cells then continue to grow at a reduced rate with a concomitant decrease in intracellular nitrogen content. Citric and isocitric acid production starts at the end of the growth phase. During about 80 hr excretion proceeds at a constant rate of 0.7 g/liter/hr for citric acid and 0.1 g/liter/hr for isocitric acid. The final citric and isocitric acid concentrations are 95 and 10g/liter, respectively. During acid excretion cellular respiration accounts for 60 and 35% of consumed oxygen and glucose. Both acid and CO₂ production rates follow a Michaelis–Menten-type dependence on oxygen concentration with Michaelis–Menten constants of 0.9 and 0.15 mg/liter for acid and CO₂ productions, respectively.     

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; )     

GROWTH AND HABITUATION IN TISSUE CULTURES OF ENGLISH IVY,HEDERA HELIX

Tissue cultures from both juvenile and adult stems of English ivy, Hedera helix L., were established in White's medium supplemented by coconut water and auxin (usually naphthalene acetic acid). With repeated transfers, cultures were habituated in less than a year to grow well without coconut water by using an auxin and kinetin. Cultures from juvenile seedlings were less demanding in requirements for growth. In all types of cultures occasionally small areas of rapidly growing cells were noticed. These when isolated gave rise to rapidly growing cultures with many cells of unusual appearance. Abnormally long cells and chain-type cells were abundant. When 0.1 mg/ liter of kinetin was added to the medium, these cells grew well without auxin and coconut water.     

Mass Propagation of Begonia x hiemalis Plantlets by Shake Culture

The conditions for rapid growth of Begonia plantlets from budsregenerated on leaf segments were studied, the ultimate aimbeing to develop a mass propagation procedure. Plantlet growthwas stimulated in submerged culture by Murashige-Skoog's mediumwith 10 or 30 g/liter sucrose, but was inhibited by a lower( x1/4) concentration of basal medium. Root development wassuperior in the lower strength medium. The greater amount ofsucrose, 30 g/liter, stimulated root growth more than the 10g/liter did. Buds required kinetin at a concentration of 0.3to 1 mg/liter, but did not require auxin. Plantlets reachedmaximum growth after 3 weeks of cultivation in 300-ml Erlenmeyerflasks containing 30 to 150 ml of medium that were shaken at180 rpm. Cultivation in jar and bubble column fermentors appearsto be advantageous for the large scale propagation of Begoniaplantlets. (Received December 8, 1980; Accepted February 4, 1981)     

Acetone and Butanol Production by Clostridium acetobutylicum in a Synthetic Medium

The effect of the component concentrations of a synthetic medium on acetone and butanol fermentation by Clostridium acetobutylicum ATCC 824 was investigated. Cell growth was dependent on the presence of Mg, Fe, and K in the medium. Mg and Mn had deleterious effects when in excess. Ammonium acetate in excess caused acid fermentation. The metabolism was composed of two phases: an acid phase and a solvent one. Low concentrations of glucose allowed the first phase only. The theoretical ratio of the conversion of glucose to solvents, which was 28 to 33%, was obtained with the following medium: MgSO₄, 50 to 200 mg/liter; MnSO₄, 0 to 20 mg/liter; KCl, 0.015 to 8 g/liter (an equivalent concentration of K⁺ was supplied in the form of KH₂PO₄ and K₂HPO₄); FeSO₄, 1 to 50 mg/liter; ammonium acetate, 1.1 to 2.2 g/liter; para-aminobenzoic acid, 1 mg/liter; biotin, 0.01 mg/liter; glucose, 20 to 60 g/liter.     

Induction of Callus from Flesh of Gardenia jasminoides ELLIS Fruit and Formation of Yellow Pigment in the Callus

Callus cultures were derived from flesh sections of young fruit of Gardenia jasminoides ELLIS on Murashige-Skoog agar medium supplemented with auxin and kinetin in the dark. Colored sections having deep yellow or orange color cells have been maintained for four years (32 generations) by subculturing on Linsmaier-Skoog Gelrite medium containing 1.0 mg/liter of indole-3-acetic acid and 0.1 mg/liter of kinetin in the dark. High-performance liquid chromatography of the yellow pigment showed the presence of crocin as a main component, but at low levels. An excess of gentiobiose, which was tentatively identified by high-performance liquid chromatography, was liberated from the yellow pigment by ammoniacal hydrolysis, but its origin other than crocin remains unknown.     

Production of Candida utilis protein from peat extracts

Sphagnum peat extracts or hydrolysates have been obtained and used as a culture medium for the production of Candida utilis biomass as single cell proteins. Acid hydrolysis of ground peat (4–60 mesh) in an autoclave operated under a set of conditions for acid strength (0.3-1.5 (v/v) H₂SO₄), holding time (1–4 hr), temperature (100–165°C), and weight ratio of dry peat to solution (3.3–16.7 g dry peat/100 g solution) yielded carbohydrate-rich extracts of different concentrations (1–34g/liter). The best yield (mg total carbohydrate/g dry peat) was obtained for a holding time of I hr and a temperature of 152°C. Low peat concentratio (4.1 g dry peat/100 g solution)resulted in high yield(280mg total carbohydrate/gdry peat) with a corresponding low carbohydrate content in hydrolysate (13 g/liter), while a lower yield with a higher carbohydrate content (34 g/liter)in hydrolysate were found when increasing peat concentration (16.7 g dry peat/100 g solution). Shake-fladk experiments using peat hydrolysates as the culture medium together with NH₄OH (～4.8 g/liter) and K₂HPO₄(5 g/liter) as nitrogen and phosphate supplement, respectively, gave a maximum biomass concentration of 7.5 g/liter after 60 hr at 30°C and 200rpm. Batch cultivation in a fermentor under controlled conditions for aeration (4.2 liter/min), agitation (500rpm), temperature (30°C), and pH (5.0) produced a maximum biomass of 10 g/liter after 20 hr with a specific growth rate of 0.13 hr^?1. For the continuous cultivation, a maximal biomass productivity of 1.24 g/gliter-he was obtained at a dilution rate of 0.125 hr ^?1. Monod's equation's equation has been used for the estimation of the coefficients μ_Max, K_s, and Y. It was found that the yield coefficient Y is not constant during the progress of batch cultivation.     

The Effective Production of Shikonin by Cultures with an Increased Cell Population

We have studied the efficient production of shikonin derivatives by suspension cultures of Lithospermum erythrorhizon with an increased cell population. The yield of shikonin derivatives was highest (800 mg/liter) when 2.8 g dry wt/liter of the cells was inoculated into the M-2 medium which we had developed for the production, but the excess inoculum lowered the yield.

We investigated suitable conditions for production with the increased cell population. The optimum amount of inoculum rose to 4.9 g dry wt/liter when the concentrations of all the components contained in the M-8 medium, which we developed for increasing the productivity by modification of the M-2 medium, were increased in proportion to the amount of inoculum, and consequently we could increase the yield of the shikonin derivatives from 1400 mg/liter to 1900 mg/liter. Moreover, the increased rate of oxygen supply in addition to the enrichment of the medium made it possible to produce 2300 mg/liter of the shikonin derivatives from a culture for which 5.6 g dry wt/liter of the cells was inoculated.     

The mode of action of a morphactin,fluoren-9-carboxylic acid

Fluoren-9-carboxylic acid acts not only as an auxin but also as an gibberellin-antagonist. In the standard pea straight test (S₅ section) for auxin it stimulated elongation, the optimum concentration being 10 mg/l. On the other hand, it inhibited elongation at 0.1 mg/l. This inhibitory effect was more marked when younger tissue (S₁ section) which also responds to gibberellin was used. Interaction of FCA and IAA in the S₅ section has shown that at higher concentration of IAA there seemed to be a suppraoptimal effect, indicating that FCA acted as an auxin. However, in the S₁ section, the stimulating effect of GA₃ was markedly inhibited by 0.1 mg/l FCA; 10 mg/l FCA was either additive or less than additive to GA₃. In the cucumber hypocotyl test FCA itself was inactive up to 100 μg/plant, but it inhibited the GA₃-induced elongation. This inhibition was overcome by increasing the dosage of GA₃. In the same material, the IAA-induced elongation was not affected by FCA. These results indicate that whether FCA acts as an auxin or a gibberellin-antagonist depends on whether the tissue is sensitive to gibberellin and/or auxin.     

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; )     

STUDIES ON THE FLORAL HISTOGENESIS AND PHYSIOLOGY OF PERILLA. III. EFFECTS OF INDOLEACETIC ACID ON THE FLOWERING OF APICAL BUDS AND EXPLANTS IN CULTURE

Raghavan , V. (Princeton U., Princeton, N. J.) Studies on the floral histogenesis and physiology of Perilla. III. Effects of indoleacetic acid on the flowering of apical buds and explants in culture. Amer. Jour. Bot. 48(10): 870–876. Illus. 1961.—The responses of apical buds and explants of a short-day plant, Perilla frutescens (L.) Britt. var. 'Tall Late,' when grown in vitro in White's medium supplemented with indoleacetic acid (IAA) and subjected to short-days (SD) or long-days (LD), are described. Additions of varying concentrations of IAA to the medium inhibited the flowering of the photoinduced apical buds in 2 ways. There was a progressive delay in the appearance of the first signs at the apex and a gradual transition from the more flower-like structures in lower concentrations of IAA (0.1 mg/liter) to sterile cones in higher doses. The sterile cones had florets with well-developed calyx and corolla lobes, but lacked the sporogenous tissues. When subjected to LD, visible signs were observed only in buds grown in 0.1 and 1.0 mg/liter IAA, the pronounced effect of the auxin being in the step-wise inhibition in the formation of the non-sporogenous tissues of the differentiating florets. Flowering of the explants with the 1st pair of unfolded leaves was also inhibited by IAA in either SD or LD, but the 1st signs appeared relatively faster than in apical buds. When photoinduced, explants with the 1st and 2nd pairs of unfolded leaves flowered in all concentrations of IAA tried (up to 100 mg/liter) while those kept in LD remained entirely vegetative.     

Role of Auxin in Induction of Polarity in Fucus vesiculosus Zygotes

We studied the effects of auxin (indole-3-acetic acid) on formation of the primary polarity axis in zygotes of the brown algae Fucus vesiculosusL. Within the first 2.5 h after fertilization, the zygotes release this phytohormone in the ambient medium. The treatment of developing zygotes with the inhibitor of indole-3-acetic acid transport from the cell 2,3,5-triiodobenzoic acid at 5 mg/l arrests the auxin secretion and leads to its accumulation in the cells. This causes a significant delay in zygote polarization. The treatment of zygotes with the exogenous indole-3-acetic acid at 1 mg/l stimulates cell polarization and formation of a rhizoid protuberance. When auxin was added to the medium with triiodobenzoic acid, the inhibitory effect of the latter was eliminated. It has been proposed that the content of indole-3-acetic acid in the ambient medium is a key factor in the induction of polarity of the F. vesiculosus zygotes.     

Formation of higher alcohols and phenol by strains of zymomonas sp.

Strains of the bacteria Zymomonas sp. were studied for their ability to form higher alcohols. In a complex growth medium, six strains were shown to produce significant amounts of 1-propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, pentanols, secondary hexyl-alcohols, and trace amounts of n-hexanol. When resting cells of these organisms were placed into a fermentation medium containing glucose and Tris-buffer, Z. mobilis 8938 produced increased levels of 1-butanol, and secondary hexyl-alcohols at concentrations of 13.5 mg/liter and 5.8 mg/liter, respectively. Another strain, Z. mobilis subsp. mobilis B 806, stimulated the formation of 1-propanol and 1-butanol at concentrations of 14.9 mg/liter and 23.52 mg/liter, respectively. Amino acids or amino acid precursors were then added to the fermentation medium. The presence of threonine and α-ketobutyric acid stimulated Z. mobilis 8938 to produce 82.6 mg/liter secondary hexyl-alcohols and 8.0 mg/liter n-hexanol, respectively. Isoleucine and valine increased the production of 2-methyl-1-butanol (394.0 mg/liter) and 3-methyl-1-butanol (113.4 mg/liter), respectively, by Z. mobilis subsp. mobilis B 806. Glutamine enhanced the formation of 2-methyl-2-butanol production to concentrations 38.8 mg/liter in Zymomonas strain B 806. Additional experiments suggested that higher alcohol production could also be accomplished in the absence of glucose when cells were allowed to metabolize the precursors only. The effect of aromatic amino acids on phenol production was determined using resting cells of Zymomonas sp. The maximum yield of phenol (111.6 mg/liter) was found by Zymomonas strain 8938 in the presence of tyrosine. The addition of phenylalanine also stimulated this strain to form 71.4 mg/liter of phenol.     

A Revised Medium for Growth of Pea Mesophyll Protoplasts

The nutrient requirements of mesophyll protoplasts from Pisum sativum L. cv. Timo have been investigated and a synthetic and completely defined medium has been designed. A high calcium concentration (12 mM) stimulated both protoplast survival and cell division. The content of iron and zinc was also critical. Additions of nicotinic acid, pyridoxine and thiamine were necessary. The protoplast growth was enhanced when some amino acids were included in the medium. An absolute requirement for auxin and cytokinin was shown. In the revised medium about 90% of the isolated protoplasts survived and formed a cell wall. The first divisions were observed after 5 days and after 1 week 10–20% of the cells had divided at least once.     

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.