GROWTH OF EXCISED PINE EMBRYOS AND THE ROLE OF THE COTYLEDONS DURING GERMINATION IN VITRO

A study was made of the role of the cotyledons, embryo orientation, surgical treatment, darkness, light, and autoclaved coconut milk on the growth of Pinus lambertiana Dougl. embryos in vitro. The embryos did not require an haustorial function of the cotyledons in vitro. Removal of the shoot meristem drastically altered the developmental physiology of the embryo and the function of the root meristem was severly inhibited. Positional effects on embryo growth were reversed by darkness. In the light vertical-inverted tube cultures displayed better growth than horizontal-inverted tube cultures, whereas in the dark the horizontal-inverted tube cultures displayed better growth than the ver ical-inverted tube cultures. Autoclaved coconut milk had no statistically demonstrable effect on embryo growth as measun d by the analysis of variance and Student-Newman-Keul's range test; however, the graphical analysis suggests that, in conjunction with the presence of the shoot meristem, there may be a slight beneficial response to autoclaved coconut milk.     

THE GROWTH AND DIFFERENTIATION OF CULTURED BARLEY EMBRYOS

Norstog , Knut . (Wittenberg U., Springfield, Ohio.) The growth and differentiation of cultured barley embryos. Amer. Jour. Bot. 48(10): 876–884. Illus. 1961.—Cultures of excised embryos of barley, Hordeum vulgare L., were made on a number of different media. Growth on White's medium was promoted by adding coconut milk. In the absence of coconut milk, amino acids did not promote growth and differentiation. Embryos as small as 60 μ were successfully grown in vitro. Smaller embryos had the capacity to initiate root and shoot primordia but did not possess the ability to form such embryonic organs as the scutellum and epiblast. Proembryos developed shoots and roots only after a period of irregular growth in which unorganized masses of cells were formed. Multiple centers of shoot initiation were observed in such embryos. The results of the study suggest that, in barley at least, embryonic form may result from an interaction between the embryo and nutritional, spatial and other factors within the ovule.     

Experimental Studies of Young Ginkgo Embryos—the Effect of Coconut Milk on the Embryos Cultured in Vitro

The present investigation deals with the effect of coconut milk upon the growth of in vitro culture of young Ginkgo embryos. The basic medium consists of White mineral salts in which Fe-citrate was used instead of Fe2(SO4)3 and vitamins (B1, 0.5 ppm, B6, 0.5 ppm, Ca-pantothenate, 0.5 ppm, niacin, 1 ppm and glycine, 2.5 ppm). 8% sucrose was used for younger embryos as the carbon source and 5% for the larger ones. The pH value of the medium was adjusted to about 6. 0.7% agar was used. The cultures were kept in an incubator (about 20–23 ℃) and no artificial light was used. The coconut milk, both filtered and autoclaved, was tested for its effect on the growth and structure of the young embryos. The important results obtained are summarized as follows: 1. The coconut milk, used both filtered and autoclaved, greatly promoted the growth and differentiation of the Ginkgo embryos cultured in vitro. 2. The optimum concentration of the coconut milk is 10%–20% for the younger embryos (900–1,600μ), while the higher concentrations (30% and over) may induce callus formation. 3. For the larger embryos the coconut milk was less effective, no callus formation occurred for the embryos over 2,800μ at isolation and for them 40% coconut milk was found more effective than 10% coconut milk. 4. There was no significant difference of the effect between the filtered and the autoclaved coconut milk. 5. From the experimental data obtained the authors conclude that the coconut milk at adequate concentration greatly promotes the rate of cell division and may initiate the meristematic regions around the shoot apex and over the whole surface of the cotyledons of the treated embryos.     

Plantlets from somatic callus tissue of the woody legume Sesbania bispinosa (Jacq.) W. F. Wight

In vitro regeneration of plantlets and multiplication of Sesbania bispinosa (Jacq.) W.F. Wight plants from cultured callus tissue were demonstrated. Callus was established from both cotyledons and mature leaflets on Murashige and Skoog (MS) basal medium supplemented with BAP (0.5 mg/l) and 2,4-D (2 mg/l). Callus mediated shoot bud differentiation was studied under defined nutritional, hormonal and cultural conditions. Various concentrations of BAP or kinetin (Kn) with coconut milk (CM) in MS media induced different levels of shoot bud differentiation as well as multiplication. Multiple shoot bud differentiation occurred in most of the primary calli. The best medium for shoot bud differentiation from cotyledon derived callus, contained BAP (2 mg/l) and 15% CM (V/V). More efficient shoot bud organogenesis was recorded with BAP than Kn. Supplementation with CM in MS media accelerated shoot bud organogenesis in differentiating callus tissue. Rooting of differentiated shoots was achieved by a three step culture procedure involving (a) MS solid medium containing IBA (2 mg/l), (b) growth regulator free half strength MS medium with 1% charcoal, and (c) half strength MS liquid medium free of vitamins, growth regulators and charcoal.Abbreviations IAA indoleacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - Kn kinetin - CM coconut milk - MS Murashige and Skoog's medium - SBI shoot bud inducing medium     

NUTRITIONAL STUDIES OF THE SUBMERGED MARINE ANGIOSPERM THALASSIA TESTUDINUM I. GROWTH RESPONSES OF AXENIC SEEDLINGS TO NITROGEN ENRICHMENT

Algae- and bacteria-free seedling cultures of the seagrass Thalassia testudinwn Banks ex König were utilized to evaluate effects of nutrient enrichment on growth and chemical composition. Seedlings cultured in media based on both synthetic seawater and NH-15 medium amended with inorganic nitrogen (NH₄⁺) and organic nitrogen (glutamine, glutamate, urea and yeast extract) exhibited no growth enhancement relative to controls in the growth parameters measured. General decreases in green leaf areas and leaf widths after one month coupled with relatively high C:N ratios after 3 months in culture suggest utilization and depletion of stored reserves with little or no assimilation of exogenous nutrients. These observations coupled with previous results in non-axenic seedling cultures indicate microbial associations may play a critical role in the nutrient physiology of this species.     

VARIATIONS WITHIN SINGLE CELL CLONES OF TOBACCO TISSUE CULTURES

Comparative studies were made utilizing two series of secondary clones (single cell clones derived from single cell clones H 196 and H 241) of hybrid tobacco (Nicotiana tabacum ♂ × Nicotiana glutinosa ♀ ) tissue grown in vitro. Secondary clones derived from a single parent varied in color, consistency, the ability to grow, and rate of growth with various carbohydrates and growth-promoting substances. The growth of the secondary clones generally resembled that of the parent clone from which derived. Many of the 23 secondary clones of H 196 grew satisfactorily on media supplemented with sucrose, dextrose, levulose, or maltose; lactose, galactose, and xylose were unsatisfactory supplements. Similarly, the series of 30 secondary clones isolated from H 241 grew well on some media but poorly on others. Growth generally decreased when α-napthaleneacetic acid or 2,4-dichlorophenoxyacetic acid was omitted from the basal coconut milk medium. Growth decreased considerably when coconut milk was omitted from the basal medium. The optimum sugar concentration was 1/2 to 1 per cent.     

Glutamine improves shoot morphogenesis in chickpea (Cicer arietinum L.)

The use of glutamine has been shown to increase the frequency of organogenesis and regeneration in the in vitro culture of several plants. The effect of glutamine on hormone-induced multiple shoot formation in desi and kabuli genotypes of chickpea (C-235 and PUSA-1053) were evaluated. Embryo axes with or without attached cotyledons were cultured in thidiazuron (TDZ) or 6-benzylaminopurine (BAP)-containing medium, respectively, with various concentrations of glutamine. Glutamine improved and prolonged the multiple shoot regeneration ability of the embryo axis. Chickpea embryo axis with attached cotyledon and cultured in TDZ-containing medium showed improved and prolonged shoot regeneration with 5 mM glutamine, while embryo axis without cotyledon and cultured in BAP-containing medium showed prolonged regeneration ability in 10 mM glutamine. Glutamine, however, did not serve as a substitute for cotyledon. Desi genotype (C-235) showed better response for multiple shoot formation as compared to the kabuli genotype (PUSA-1053). Glutamine at a concentration of 5 mM also improved root formation in excised in vitro shoots.     

GROWTH INDUCTION IN CULTURES OF HAPLOPAPPUS GRACILIS. I. THE BEHAVIOR OF THE CULTURED CELLS

Blakely , L. M., and F. C. Steward . (Cornell U., Ithaca, New York.) Growth induction in cultures of Haplopappus gracilis. I. The behavior of the cultured cells. Amer. Jour. Bot. 48(5): 351–358. Illus. 1961.—Because of the unusual cytology of Haplopappus gracilis (2n = 4), a study has been made of the growth of its stem tissue in culture. Although growth may occur on a basal medium supplemented in various ways, it was stimulated for present purposes by the use of a basal medium containing casein hydrolysate, coconut milk (2–10% by volume) and naphthaleneacetic acid (0.5 p.p.m.). A definite synergism between coconut milk and naphthaleneacetic acid was demonstrated. The general form of the colonies so obtained responds to the composition of the medium, and certain effects of pigmentation indicate that the biochemistry of the cultured tissue is also a function of the conditions. The Haplopappus cultures were maintained in liquid culture either in the form of free cells or of the small cell clusters to which they readily gave rise. The form of typical cells and cell clusters is described, and stress is laid upon the range of growth forms that are encountered. Variations in suspensions of cells, or small cell clusters, may be investigated by the application of simple microbiological techniques. Haplopappus gracilis is thus a useful material for the further study of growth and morphogenesis by tissue culture techniques.     

Regulation of seed germination and polarity in seedling development inOrobanche aegyptiaca by growth substances

InOrobanche aegyptiaca PEES. (Orobanchaceae) the mature seed is tiny and contains a subglobose embryo which is not differentiated into radicle, hypocotyl, plumule, and cotyledons. In aseptic seed cultures on medium TB supplemented with yeast extract or coconut milk, both roots and shoot originated from the morphological radicular pole of the embryo (monopolar pattern). The bipolar mode of seedling formation, that is a shoot originating from the plumular pole and roots from the radicular pole, ensued on the basal medium THS and on TB supplemented with certain concentrations of IAA, kinetin, GA3, or strigol.     

SEEDLING GROWTH STUDIES OF EARLY-RIPENING PEACHES. I. INTERRELATIONSHIP BETWEEN EMBRYO MATURITY,GROWTH SUBSTANCES AND SEEDLING GROWTH

Weaver , Gerald M., and L. Fredric Hough . (Rutgers, The State Univ., New Brunswick, N. J.) Seedling growth studies of early-ripening peaches. I. Interrelationship between embryo maturity, growth substances and seedling growth. Amer. Jour. Bot. 46(10): 718–724. Illus. 1959.—Peach fruits of ‘Raritan Rose’ were harvested 75, 83, 91, 99 and 107 days after full bloom. Embryos were excised, and cultured on artificial media. Growth responses following 8 wk. cold treatment varied considerably with embryo maturity. No correlation existed between seedling survival and the initial responses of shoots. Root growth of 83-day embryos exceeded that of all other stages of maturity. This was reflected in maximum seedling survival and vigor. Initial shoot growth was greatest from embryos harvested at 91 days and at maturity (107 days), but all shoots developed into rosettes which may expand slightly or develop normally from axillary buds. Bioassays of growth substances suggest a strong correlation between early shoot development and auxin concentration. Root growth-responses appeared to be correlated with a promotor-inhibitor balance. The results of wheat coleoptile assays of embryo extracts chromatographed in an isopropanol: ammonia: water solvent (8:1:1) suggest the presence of three growth promotors, in greatest concentration at Rf 0.70, but also at Rf values 0.10 and 0.30. Two growth inhibitors were present in embryo tissue at Rf values 0.27 and 0.96, the former decreasing in concentration with increasing embryo maturity.     

THE PHOTOINHIBITION OF GROWTH IN ETIOLATED STEM SEGMENTS. II. GROWTH CAUSED BY COBALT IN PISUM

Bertsch, Walter F. (Yale U., New Haven, Conn.) The photoinhibition of growth in etiolated stem segments. II. Growth caused by cobalt in Pisum. Amer. Jour. Bot. 50(3): 213–219. Illus. 1963.—Etiolated pea stem sections were utilized to study the effects of various metal ions on the red, far-red photoinhibition of stem growth. Excision of immature tissue results in loss of photosensitivity. All growth caused by cobalt, either in the presence or absence of sugar, was inhibited by brief exposure of the excised tissue to red light. Since growth caused by sugar is also light-sensitive, it may be said that either cobalt or sugar may induce photosensitivity in this immature tissue. Other divalent metal ions were tested, but only nickel promoted growth. Although cobalt and nickel showed the same optimal concentration (ca. 2 × 10⁻⁵ m), growth caused by nickel was not photosensitive, suggesting that cobalt and nickel promoted growth through separate mechanisms. Since growth caused by indole-3-acetic acid or gibberellic acid is also insensitive to light in this tissue, a photosensitive sugar-cobalt growth system has been experimentally separated from other growth-controlling systems. The photoinhibition of a growth system which utilizes sugars and is promoted by cobalt and the relation of this system to certain de-etiolation phenomena are discussed.     

An analysis of seed development in Pisum sativum. VII. Embryo development and precocious germination in vitro

Three different culture media have been examined for their ability to support growth in culture of embryos of two pea lines near-isogenic except for the r-locus. Embryos showed a greater increase in fresh weight on a medium containing 10% sucrose and a high level of a mixture of amino acids than on either one containing an equivalent amount of glutamine as the sole nitrogen source or one containing both inorganic nitrogen and a low level of glutamine. Small embryos (up to 10 mg fresh weight) showed the greatest relative increase in fresh weight. Decreasing the osmotic pressure of an agar medium by lowering the sucrose content to 2% and reducing the concentration of amino acids induced precocious germination. Shoot growth was more sensitive than root growth to increasing sucrose concentrations and optimum development was obtained when embryos were cultured in liquid culture at a high osmotic pressure followed by growth on an agar medium at low osmotic pressure. Alternatively, precocious germination could be induced by removing the cotyledons. Embryos of all sizes and of both genotypes of pea responded in a similar manner.     

Metabolism of Urtica dioica as dependent on the supply of mineral nutrients

Plants of Urtica dioica L., a very nitrophilous species, were grown in a nutrient solution containing either high (100%) or low (2%) nutrient supply. Part of these plants were subjected to a sudden switch from 100% to 2% or vice versa. Plant weight, sugar and organic nitrogen (both soluble and insoluble) and nitrate content were measured during growth. The activities of two nitrogen assimilating enzymes, nitrate reductase (NR) and glutamine synthetase (GS) were determined.
Growth of Urtica dioica was retarded at low nutrient supply. Root growth was limited by another factor than nitrogen. This was shown by a higher protein content. In the first period after a switch from 100% to 2%, redistribution of nitrogen from shoot to root could be demonstrated, and leakage from the root into the nutrient solution. It is suggested that in these conditions GS in the root reacted to this downward flux. Comparison with earlier findings on the less nitrophilous Plantago lanceolata showed that at 100% nutrient supply a correlation occurs between nitrate reduction and glutamine synthetase activity in that plant part which exported reduced nitrogen: the root in P. lanceolata and the shoot in U. dioica. In the importing plant part, glutamine synthetase was influenced by nitrate reduction as well as by imported reduced nitrogen.     

香菇菌丝深层培养的碳、氮源和生长因子

本文分别设计了生长刺激因子、碳、氮源试验基本培养基。结果表明在深层培养时,废糖蜜、粗制蔗糖、玉米浆、酵母粉、麸皮浸汁中含有刺激香菇菌丝生长的物质。而各种维生素(包括 B_1)、氨基酸、有机酸、植物生长激素等均无明显的作用。在上述生长因子的促进下,香菇菌丝可利用无机或有机氮源。无机氮源以 NH_4NO_3,最好,其次为(NH_3)H_2PO_4、NH)4Cl 等。有机氮源以玉米浆、酵母粉最好。碳源以玉米淀粉最好。本文并介绍了一种廉价的、良好的香菇菌丝液体培养配方,可以稳定地培养出细小均匀的菌丝球、生长丰满稠密的液体菌种。     

Prolific shoot regeneration from immature embryo explants of sainfoin (Onobrychis viciifolia Scop.)

Summary Immature cotyledons and embryo axes of sainfoin were cultured on Murashige and Skoog (MS) media supplemented with various concentrations of 6-benzylaminopurine (BAP) and a-naphthaleneacetic acid (NAA) to induce adventitious shoot regeneration. The highest frequency of shoot regeneration occurred following an initial callus growth on a MS medium containing 0.5 mg/l BAP and 2 mg/l NAA. Immature embryo axes showed higher regeneration capacity than immature cotyledons, however, shoot elongation was best achieved on immature cotyledons. Regenerated shoots were excised and rooted in half strength MS medium with 1 mg/l indole-butyric acid (IBA) or 1 mg/l NAA. The rooted plantlets were finally transferred to compost.     

THE INFLUENCE OF GROWTH SUBSTANCES ON THE INDUCTION OF APOGAMY IN PTERIDIUM GAMETOPHYTES

Experiments employing sequences of three media demonstrated the effect of growth substances on the induction of apogamy. The most effective sequence was 4% sucrose 1–14 days, 4% sucrose plus growth substance 15–28 days, and 0.1% sucrose 29–56 days. In this sequence concentrations of NAA, IAA, and GA promoted apogamous shoot formation. A higher NAA concentration than optimal for shoot formation stimulated apogamous root formation in all medium sequences. Kinetin was without effect or inhibitory to apogamy. Combination of kinetin/GA or GA/NAA concentrations did not increase the apogamous response. One combination of the kinetin/NAA concentrations had a synergistic effect on the apogamous shoot formation. Additions of GA to the synergistic kinetin/NAA combination had an antagonistic effect on the apogamous response.     

GROWTH OF EDIBLE CHLOROPHYLLOUS PLANT TISSUES IN VITRO

Hildebrandt, A. C, J. C. Wilmar, H. Johns, and A. J. Riker. (U. Wisconsin, Madison.) Growth of edible chlorophyllous plant tissues in vitro, Amer. Jour. Bot. 50(3): 248–254. Illus. 1963.—Plant callus cultures were attempted from roots, stems, leaves or excised embryos of 32 species of plants on a basal mineral salts–sucrose agar medium (T-medium), on T-medium + coconut milk + α-naphthaleneacetic acid + calcium pantothenate (C-medium) and on C-medium + 2,4-dichlorophenoxyacetic acid (D-medium). Embryos on T- or C-medium generally produced normal plants, while on D-medium, they often produced callus only. Fresh isolates of carrot, endive, lettuce, parsley, red kidney bean, and navy bean gave moderate to excellent callus on C-medium. Parsley and navy bean also produced excellent callus on D-medium. Strains of callus from potato, tomato, grape and rose also grew well on C- or D-medium. In the light, red pigmentation developed on rose, parsley, and grape callus. Chlorophyll formation was inhibited on D-medium, but on C-medium more or less chlorophyll was initiated in callus from carrot, endive, lettuce, pea, potato and certain rose varieties. Chlorophyll formation was also strong in endive callus on T-medium supplemented with casein hydrolysate, i-inositol and NAA. The amount and type of sugar used in C-medium influenced the amount of growth and were critical in relation to chlorophyll formation. Carrot tissues in constant light produced abundant chlorophyll and were still growing on media without added sugar after 6 weeks.     

CLONAL VARIATION OF GRAPE-STEM AND PHYLLOXERA-GALL CALLUS GROWING IN VITRO IN DIFFERENT CONCENTRATIONS OF SUGARS

Arya , H. C., A. C. Hildebrandt , and A. J. Riker . (University of Wisconsin, Madison, Wisconsin.) Clonal variation of grape-stem and Phylloxera-gall callus growing in vitro in different concentrations of sugars. Amer. Jour. Bot. 49(4): 368–372. Illus. 1962.—The original callus grown from normal tissue and that grown from gall tissue contained mixtures of different kinds of cells. To study the variability, a large number of clones were developed by single-cell technique. From these, 6 clones were selected for detailed study. Growth was compared of 6 single-cell clones established in vitro; 3 from normal grape stem and 3 from gall tissues incited by Phylloxera vastatrix Planch. The clones were stable in growth rate (fast, medium, and slow) when grown on modified White's basal medium supplemented with coconut milk, α-naphthaleneacetic acid (NAA), and calcium pantothenate. Growth was measured after 6 weeks as the average wet weight on concentrations from 0.06–4.0% of sucrose, d(+) dextrose, d(-) levulose, d(+) mannose, d(+) galactose, and d(+) lactose, respectively. Every sugar, except mannose, was a suitable source of carbon. The cells were not all alike in their growth response to different sugars. Gall- and normal-tissue clones grew best with 0.125% sucrose, levulose, and galactose. With dextrose and lactose, optimum yields were obtained at the 1.0% sugar level. Growth of fast-, medium-, and slow-growing clones was altered with the type of sugar. Gall and normal tissues were differentiated from each other when grown on mannose in which gall tissues grew best at 0.125% and normal at 1.0% levels. Gall tissues as a group were able to grow better with mannose than the corresponding normal ones. Levulose, on the other hand, favored growth of normal clones in comparison to that of diseased ones. Sugars varied in their inhibitory influence at the 4.0% level. Dextrose, levulose, and lactose at 1.0% proved better than sucrose for the growth of all except one fast-growing gall clone which grew best with sucrose. However, at 0.125% sugar levels, even in cases where high yields were obtained, the physical character of the tissues changed to dry, brownish, and very friable. Thus, the original callus from normal and gall tissues contained cells with diverse characteristics. The various clones developed used the same sugars but varied strikingly in the rate and type of growth on certain sugars.     

MORPHOGENETIC STUDIES ON HAWORTHIA: ESTABLISHMENT OF TISSUE CULTURE AND CONTROL OF DIFFERENTIATION

Investigations were carried out on the in vitro morphogenetic responses of inflorescence segments and gynoecia of several species of Haworthia (Liliaceae). Morphogenetic responses of explants were not species specific. It was found that coconut milk was essential for the growth and differentiation of Haworthia tissue if White's basal medium was used. However, growth and differentiation could be supported by a modified Murashige and Skoog's medium, without any supplements. The investigations demonstrated’ the importance of inositol and ammonium nitrogen in the nutrition of Haworthia tissue cultures. A chemical control of callusing and shoot and root differentiation was obtained by providing appropriate amounts of auxin and cytokinin in the culture medium.     

CULTURE OF MATURE ECOTYLEDONOUS EMBRYOS OF PHASEOLUS VULGARIS AND THE NUTRITIONAL ROLE OF COTYLEDONS

Embryos of Phaseolus vulgaris L. were excised from seeds and cultured with cotyledons removed to determine the actions of various cultural conditions upon embryo development. Four media were tested, but ecotyledonized embryos did not grow as rapidly on any of them as did embryos with intact cotyledons on agar-water media. Comparisons of growth of ecotyledonized embryos with embryos bearing fractions of cotyledons indicated ecotyledonized embryos cultured on nutrient media grew about as well as embryos bearing cotyledons from which 97% of the volume had been removed surgically. The final weight of ecotyledonized embryos was greater when detached cotyledons were placed near them and was even greater when extracts of detached and incubated cotyledons were employed in the nutrient medium. Benzyladenine, kinetin, gibberellic acid, indole-acetic acid, presence of sucrose, and light or dark culture failed to enhance the ability of incubated cotyledons to stimulate growth of embryos.