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This paper described the changes in the free and bound GA-like substances in flower initiated and vegetative plants of Aster savatieri grown for 5 weeks at a temperature between 4 and 5°C or between 20 and 25°C.The GA-like substances were extracted by a modified JONES′ method. Extracts were prepared with aqueous solvent after separating a plant into three parts; shoot apex region with younger leaves, rosetted stem and older rosette leaves, using 120 plants. After fractionation, ascending paper chromatography on Toyo No. 51 was carried out using 80% isopropanol as the solvent. Bioassay methods for comparing the activity of GA-like substances in the eluate from different zones of chromatograms were the rice endosperm (Oryza sativa cv. Kotaketamanishiki) test and the rice seedling (cv. Tanginbozu) test. Results were as follows:The free and bound forms of GA-like substances were present in this plant.The shoot apex region and rosetted stem in cold treated plants showed an increase of GA-like substances in the free form and a significant decrease in the bound form. While these parts in non-cold treated plants showed a decrease in the free form and an increase in the bound form. Similar tendencies were observed in both flower initiated and vegetative plants. However, the concentrations of GA-like substances in flower initiated plants changed more greatly than those in vegetative ones during the low temperature treatment. Changes in the activity of GA-like substances in older rosette leaves were not great as in shoot apex region and rosetted stem.Changes in the neutral GA-like substances in plants did not occur during the treatment.The data indicated that the low temperature caused an increase of the free GA-like substances which played an important role on the rapid stem elongation.

Maize caryopses were exposed in moist, sterile sand to temperatures of 24°, 10° and 6°. The amino acid content of embryo, endosperm and pericarp tissues of kernels and amino acid occurence in sand eluates was analysed by paper chromatography.

The phospholipid-glycolipid mixture, isolated chromatographically from the “free” (hexane soluble) and “bound” (hexane insoluble, water-saturated butanol extractable) lipid of wheat endosperm, was fractionated by column and thinlayer silicic acid chromatography. The components were identified by hydrolysis followed by thin-layer or paper chromatography of the products. They included a sterol-containing glycolipid, hitherto unreported in wheat. The fatty acid and sterol compositions of the phospholipidglycolipid components were determined by gasliquid chromatography. Differences were found between varieties and between components.

1. Free amino acids and amino acids yielded by acid hydrolysis of separate samples of endosperm and embryos of ten lines of maize were measured by paper chromatography. 2. A similar distribution was found among the lines tested with regard to both kind and concentration of amino acid present.

Changes in soluble sugar content in individual parts (embryo, endosperm and pericarp) of maize caryopses exposed to the influence of 24°, 10° and 6° were studied. At the same time the eluates from sand in which the seeds were planted and exposed for various periods were analysed by paper chromatography.

1. This paper deals with the behavior of seeds of Magnolia grandiflora during preparation for germination and the early stages of the process, with particular reference to the overcoming of delayed germination, to the chemical changes taking place during after-ripening, and to the presence and development of oxidizing mechanisms. 2. Dry storage in the pulp at room temperature seemed almost as advantageous as moist storage of the disinfected seeds at 10⚬C. 3. Seeds with the fleshy outer coat retained when set to germinate were destroyed by fungi. It was necessary to remove this coat and soak the seeds in CaOCl2 solution in order to control this situation. 4. The lignified coat delays germination but does not prevent it in 5-20 per cent of the seeds. Alternation of temperature was the only means found of securing consistent germination percentages above the one just mentioned. 5. During the preparation for germination the seeds acquire an increased resistance to fungal attack. 6. The endosperm of a dry seed...

UNDERSTANDING the metabolism of the developing maize endosperm is a problem of practical and theoretical importance. Carbohydrate metabolism in the maize endosperm has been investigated extensively by means of various biochemical and genetic techniques (Culpepper and Magoon, 1924, 1927; Bernstein, 1943; Sprague et aL, 1943; Andrew et al., 1944; Cameron, 1947; Kramer and Whistler, 1949), but nitrogen metabolism of the endosperm has remained relatively unexplored. Zeleny (1935) and Watson (1949) have shown that endosperm proteins (largely zein and glutelin) are synthesized during the entire kernel maturation period, but that most of the glutelin (alkali-soluble) proteins are synthesized during the first half, and most of the zein complex of proteins (alcohol-soluble) during the last half of the endosperm maturation period. Biochemical genetics studies (Showalter and Carr, 1922; Frey et al., 1949; Frey, 1951) and analyses of various varieties and genotypes of maize endosperm (Hansen et al., 1945) show that there is a direct correlation between the content of zein and of total protein in the endosperm; this mav indicate that alcohol-soluble proteins are the major storage proteins of the endosperm. Interrelations of tryptophan, niacin and indoleacetic acid in the developing maize endosperm have been investigated by Stehsel and Wildman (1950). Their studies are perhaps the first attempt to explore the nitrogen metabolism of the developing maize endosperm on the level of individual enzymesubstrate-product reactions. It is only on this level that one can investigate adequately the various metabolic processes of the developing endosperm. Thus, a working knowledge of the kinds and amounts of nitrogenous components in the endosperm throughout its development is fundamental to understanding endosperm protein synthesis. This study is a preliminary investigation of the free amino acids and amides of the developing endosperm of four varieties of corn. Partition paper chromatography, an analytical technique proven to be useful for rapid surveys of kinds and approximate amounts of amino acids in other plant tissues (Dent et al., 1947) has been adapted for use in this investigation. MATERIALS AND METHODS.-Four morphologically dissimilar varieties of maize: Longfellow Flint

Cotyledons of germinating sunflower, pumpkin, linseed, and watermelon seeds and the endosperm of germinating castor bean seeds have been examined for their ability to utilize glyoxylate-C14and acetate-C14for the biosynthesis of amino acids. All of the tissues examined readily utilized these acids when supplied in micromolar amounts to tissue slices. The chief products of this utilization included the organic acids of the glyoxylate and tricarboxylic acid cycles and a number of amino acids and amides. The results are interpreted as indicating that, in sunflower, watermelon, linseed, and pumpkin seeds, malate formed in the malate synthetase reaction is metabolized by the partial reactions of the tricarboxylic acid cycle. α-Ketoglutarate produced by these reactions is extensively utilized in the biosynthesis of glutamate, γ-aminobutyrate, and glutamine. In agreement with data already published, castor bean endosperm utilized acetate for the biosynthesis of sugars. This tissue also utilized glyoxylate for the formation of glycine, serine, glycollate, and malate. It is concluded that, with the exception of castor bean endosperm, acetyl CoA arising as a result of fatty acid oxidation might be utilized for amino acid biosynthesis via the partial reactions of the glyoxylate and tricarboxylic acid cycles.

Stowe and Thimann1 reported that they had isolated indolepyruvic acid from maize endosperm by paper chromatography. They used the methods of ammoniacal chromatography. However, Bently2 found that under these conditions synthetic indolepyruvic acid breaks down into a number of compounds. Moreover, in a recent paper by Kaper and Veldstra3, it was shown that both synthetic and isolated indolepyruvic acid break down when chromatographed in a basic solvent. Srivastava4 has also obtained results similar to that reported by Kaper and Veldstra.

1. 1. Particulate fractions from mid-log phase suspension cultures of Lolium multiflorum endosperm incorporate [ 14 C]glucose from UDP[U- 14 C]glucose into polymeric products and into unidentified compounds soluble in chloroform-methanol and 66% aqueous ethanol. 2. 2. The fraction from the cell homogenate sedimenting at 10 000–250 000 × g was most active in polysaccharide synthesis. 3. 3. Glucose was the only radioactive sugar detected in the high molecular weight product. 4. 4. The product was shown to be a mixture of β-glucans by fractionation on the basis of solubility in water and 1 M NaOH and from the pattern of products released by three β-glucan hydrolases with different substrate specificities for β-1, 3-, β-1, 4- and β-1, 3; 1,4-glucans. 5. 5. The proportion of 1,3- and 1,4-linkages in the total products was shown to vary with the concentration of UDPG. At 1 μM UDPG 90% of the residues in the total product were 1,4-linked, whereas the proportion fell to 49% at 1 mM UDPG. 6. 6. The water soluble polsaccharide which is the predominant product at 100 μM UDPG was examined by enzymic hydrolysis and shown to be a β-1,3;1,4-glucan similar to the glucan present in the Lolium endosperm cell walls. 7. 7. The UDPG concentration in the cells during growth has been measured. It reaches a maximum of 30 μM at mid-log phase.

γ-AMINO-BUTYRIC acid has been described as present in the aqueous and alcoholic extracts of a number of plant and animal tissues1–7. In the course of studies on the nitrogenous constituents of coconut (Cocos nucifera) endosperm using paper chromatography, I have found this amino-acid to be present in amounts apparently larger than any other amino-acid except alanine in the watery liquid inside the mature fruit. Further investigations at three consecutive stages of development, namely, 4–5 months, 5–6 months and 6–7 months after setting of the fruit revealed that γ-amino-butyric acid is not present in the liquid endosperm at 4–5 months after setting of the fruit, but makes its appearance in the liquid when the solid endosperm has been deposited as a thin gelatinous layer on the nucellus in the fifth to sixth month after setting.

1. The object of the study was the isolation of highly polymerized DNA from a plant source. 2. Maize kernels in the milk stage were chosen as a source because of the free nucleate stage of the triploid endosperm and the absence of chlorophyll. 3. A method, based on phenol treatment in the presence of a NaCl-sodium dodecylsulphate-Tris-EDTA buffer (pH 9.0) is described for isolating DNA. 4. Ultracentrifugation and viscosity show that the DNA can be isolated in different sizes, depending on the extraction procedure. 5. The sedimentation coefficient, at zero concentration, of the largest molecules agree well with those of certain bacteria and bacteriophages.

Three isozyme variants of catechol oxidase have been shown to be determined by alleles of a gene, Cx, which has been located on chromosome 10 less than 0.1 recombination units from the endosperm marker du 1.—The extractable form of the enzyme is modified by an endogeneous "modifier" which appears to function as an enzyme substrate. Enzyme and modifier are functionally isolated in intact cells. Modified enzyme has altered kinetics, does not migrate in electrophoresis and most probably results from a "tanning" of the enzyme by reaction products. The content of modifier varies in different lines and is genetically determined by gene(s) independent of Cx. Treatment with maleic hydrazide causes a ten-fold reduction in the modifier content of seedlings, allowing the enzyme to be extracted in an unmodified form which will migrate in electrophoresis.—This system of enzyme and modifier fits the requirements of hypersensitive disease resistance in plants and may provide a test system to investigate the biochemical basis of disease resistance.

The seed-feeding bug Lygaeus equestris changes and extends its food plant spectrum during its life-cycle. Whether this is the result of only the natural succession of the food plants, or also includes a change in the insect's food preference during its life, was the primary question of the present paper. Feeding-choice tests using ripe seeds from important food plants in the laboratory showed that Cynanchum vincetoxicum seeds were preferred during all stages of life. Among the lesser preferred seeds tested, a shift in preference occurred during the bug's development. It seems probable that this shift may be due as much to the physical as to the chemical properties of the seeds. It is suggested that most of the other plant species exploited for feeding by L. equestris probably serve mainly as substitute food when suitable developmental stages of C. vincetoxicum are absent or in short supply. Furthermore the paper gives some notes on duration of feeding upon seeds and effects of seed coat at initiation of feeding on Cynanchum and Helianthus seeds. During the third larval instar, the preference switches from the endosperm to the seed coat for initiation of feeding on Cynanchum seeds, and so it remains during the following stages. On Helianthus seeds, only adult L. equestris feed as willingly through the coat as directly from the endosperm.

THE FIRST PAPER of this series described the development of Dianthus chinensis L. from the origin of the ovule primordium to the mature seed. The processes involved in seed production require a continual supply of nutrients from translocation or from storage tissue-s which may sometimes be identified in part as visible reserves within ovary, ovule, fruit and seed, or at others may be used currently in soluble form without temporary storage and consequently may be detected only by special tests. In the development of ovary and ovule to fruit and seed, certain tissues and cells are concerned with the maintenance of an adequate supply of nutrients. These provisions include: (1) conducting tissues through which water from the soil and soluble foods from other parts of the plant are moved, (2) absorbing cells and tissues of embryo sac and embryo that utilize the major portion of food thus accumulated, and (3) various regions of ovary, ovule. fruit and seed in which food reserves are deposited either temporarily or permanently. There are few accounts of seed development in the literature in which the accumulation and depletion of food reserves have been traced from the origin of the ovule to seed maturity. Much more frequent are reports of haustoria or other evidences of absorption and digestion associated with pollen tube, embryo sac, embryo and endosperm. The present paper deals with these aspects of nutrition in the seed of Dianthus chinensis, particularly the correlation of starch deposits in ovary, ovule, fruit and seed with the phases of ovule and embryo growth. Material used in general observations was prepared as described in the previous paper (Buell, 1952). For determining starch distribution in ovary and ovules, free-hand sections of fresh material were placed in weak 12KI solution. Since it was found that paraffin sections treated with 12KI gave results similar to those with fresh material, such preparations were used extensively because of the greater clarity of detail possible in thinner sections. Brief washing of iodine treated sections with 75 per cent sulphuric acid intensified the color of the starch grains and produced a clear blue reaction on cellulose cell walls. POSSIBLE PATHWAYS FOR TRANSLOCATION.-The vascular supply in the ovule begins as a provascular (procambial) strand differentiated in the funiculus of the ovule primordium. Even the youngest pro

When barley grains are germinated in absence of external supplies of nitrogen, their metabolism of amino-acids and proteins is dominated by the transformation of reserves, stored in the endosperm, into protoplasmic proteins and other nitrogenous constituents of the young embryo. In an earlier paper of this series (Folkes, Willis and Yemm, I952), a general account was given of this movement of nitrogen into the embryo, and more recently fairly complete analyses of the amino-acid content of the proteins of the endosperm and embryo have been described (Yemm and Folkes, I953; Folkes and Yemm, I956). It was evident from the wide differences in composition between the chief storage proteins of the endosperm and the protoplasmic proteins of the embryo that an extensive interconversion of amino-acids must occur during germination. In the present paper data are given which permit a more exact estimate of the nature and extent of these interconversions. Methods have been developed whereby barley seedlings could be analysed at various stages of germination and estimates made of the amounts of the protein fractions whose amino-acid composition has already been described. The results of these estimations, taken together with analyses of the nonprotein constituents, yield a fairly complete account of the quantitative changes in the various amino-acids during germination. The interconversions thus revealed have been considered in terms of the mechanisms involved and in relation to the respiratory process of the seedlings.

Summary. 1. The methods used in these investigations into the problems of absorption of water by seeds are described. A modification in the method of direct weighing is suggested. 2. The role of the pale in absorption is discussed. The pale exercises a retarding effect on water absorption, so long as it is attached to the caryopsis. But the attachment is broken as the absorption of water with consequent swelling extends from base to apex. 3. The cuticular membranes of the seed retard the absorption of water, but the permeability is increased by the stretching of the cuticle, consequent upon swelling. 4. The earliest absorption takes place through the micropyle. This water can diffuse upwards to a certain extent. At the higher level it causes the endosperm to swell, which in its turn induces greater permeability of the cuticular membranes. The water absorbed at this higher level diffuses upwards to a slight extent, and causes greater permeability at a still higher level. Thus, there is a progressive extension upwards of the area of absorption induced by the slight upward diffusion of water. The writer wishes to acknowledge his indebtedness to Prof. W. Stiles for help in the preparation of the manuscript of this paper; to Mr F. R. Horne for help in the course of the work; and to Mr R. L. Stevenson for making the diagrams which accompany this paper.

The formation of the vermiform appendage of the endosperm from the lower end of the embryo-sac in the seeds of Grevillea robusta cunn., a member belonging to the proteacem, has already been mentioned in a previous paper published some time back (Kausik, 1938). Recently, a further detailed investigation of this remarkable stucture was made with a view to determine the series of stages in its origin, growth and final fate during the development of the seed. A brief account of some of the outstanding features met with in this development is given in the following note, the details being reserved for a fuller paper to be published shortly.

THE ANATOMY of the tribes Malveae and Ureneae of the family Malvaceae was reported in an earlier paper (Reeves, 1936). The work being reported in the present paper deals with the seeds of the tribe Hibisceae. The sequence of genera included here in general is that of Schumann (1895), and the species of Gossypium are arranged according to Watt (1907). The classification of the cottons proposed by Zaitzev (1927-1928), which in some respects seems superior to all others, could not be used because of its incompleteness. This report is a continuation of the earlier paper, and the methods used are the same. PARITIUM TILIACEUM Juss.-Seeds of Paritium tiliaceum were sent to the writer from Coconut Grove, Florida, by Mr. H. F. Loomis of the Bureau of Plant Industry, U. S. Department of Agriculture. The seed is campylotropous, but its cuirvature is only a little more than 45 degrees (fig. 29). It is dark brown in color and bears numerous short, brown epidermal hairs. Stages of development were not studied, and therefore the tissues arising from each integument were not determined. The internal structure of the mature seed, however, is similar to that of Abelmoschus esculentus. The palisade cells are covered by one or two layers of cells which are shriveled and often fragmentary (fig. 30). The palisade cells themselves are similar to those of Gossypium hirsutum, except that they are generally smaller. The light line is conspicuous. The fragmentary remains of the protoplast and the markings on the cell walls a-re also similar to those of Gossypium hirsutum. Below the palisade tissue is a pigmented tissue. Its cells have thick walls and their lumina are filled with a substance identical in appearance with that found in the pigment layers of the genera described heretofore in this paper. The cells in the pigmented layer may or may not be compressed. The next tissue is the fringe tissue with peculiar markings on its cell walls (fig. 31). The cells of this tissue also are brown in color. Closely attached to it are the remains of the endosperm, which usually consist of a single row of cells. In the embryo the folding of the cotyledons is essentially like that of Malvaviscus drummondii. Crystals which are soluble in hydrochloric acid but insoluble in acetic acid, sulphuric acid, and xylene are very abtundant in the mesophyll tissues of the cotyledons. Results of the other microchemical tests conducted on the seed of this species indicate that the epidermal hairs are composed largely of lignin but partially of cellulose. The epidermis seems to be composed chiefly

The purpose of this report is to explain some of these Early growth of the com seedling was studied in relaenergy relationships involved in the germinating seed. tion to its dependence on energy from endosperm and early photosynthetic tissue. ~easurc:ments ,..ere made of EXPERIMENTAL PROCEDURE (1) endosperm utilization rate of seedlings grown in light and dark; (2) growth and leaf area of seedlings as affectEndosperm Utilization and Growth of Com ed by partial endosperm removal; and (J) photosynthesis (P), respiration (R), and .':CO, up~ake of seedlings as Hybrid field com seed was selected because of its large size. affected by endosperm eXCISion at different ages. Endo'Ve determined endosperm utilization and growth of corn seedsperm utilization and growth rates were similar in l~ght lings in the light and in the dark in t\\-O experiments. In the and dark unt.il the two.leaf stage 10 days after germmafirst experiment, 28 \..eighed lots of 10 seeds each were germintion. Regression of root and shoot growth on age was ated in the light and 28 similar lots in the dark. Seedlings were 15.4 mg/day and of root and sh~t growth on endosperm grown on plexiglass slant boards covered \vith \vet blotter paper. ,,-eight loss was .65 mg/mg. Partial removal of the endoSeeds from each lot \vere placed in a line 7.6 cm from the top sperm at seeding resulted in decreased growth of roots of the board and held in place by wetted tissue paper. The and shoots and indecreased leaf area per seedling. Apseeds ,vere then covered "ith blotter paper, \vhich remained parent P was uegati~'e at 9 days of age and was greatest \"et from \"ater mo\ing up the boards from trays set at their for seedlings "ith endosperm excised. Apparent P was bases, The plants \"ere grown in a growth chamber with a positive and similar at days 11, 12, and 13 for seedlings temperature of 21,8 :t: 1 C. a light intensity of 20,000 lux and "ith endosperm e.xcised and was positive, and increased a photoperiod of 15 hours. For the dark treatment. slant boards from day 11 to day 13 for seedlings with endosperm. ,vere covered with a large cardboard box. Temperature within Energy obtained from CO, fixation was not equivalent the box was assumed to be equal to that of the growth chamber. to that supplied by the endosperm until the lOth day. At Two seed lots ,vere sampled from both light and dark treat. this time seedlings had two fully emerged leaves. Absorpments during a 22-day period at 2-day intervals from germination of 1'CO, ',-as similar for seedlings \vith and without tion. At each sampling date, seedlings ,vere separated into endospetm, and increased ,,'ith age as a result of increased roots, shoots, endosperm, and scutellum. These components leaf area and increasl'{l absorption per unit of leaf area. \vere placed into ,veighing bottles, oven-dried. and weighed. Endosperm removal resulted in greater translocation of The second study \vas similar to the first, except that sam. 1'CO. to seedling roots. plillg \vas at I-day intervals from the 5th throllgh the 16th day, . .. and the sclltellum ,''as weighed \vith the endosperm. Additional inde_'\: words: PhotosynthesIS, Respiration, Gennination energ,.. . Photosynthesis and Respiration Corn seedlings \..ere gro\vn on slant boards in the light under S OURCES of energy for early growth of a grass seedthe experimental conditions previously described. From the 8th ling are: first, the endosperm; second, the endoto the 12th day, the largest and most unifo,rm seedlings w.ere Sperm and ne" leaf material' and third new leaf mas~lected and paired for study of photosynthetic, (P) and resplra. . . , h tlon (R) rates. From one member of each pair the endosperm tenal after depletion of the endospe~n. T ese stages \vas excised \vith a razor blade. Each seedling was then placed of energy dependence have been denned by Whalley. in a 65-cc bottle half-filled with a complete nutrient solution, '~ McKell, and G;reen (6) as hete;otrophic, transitional, and ,vas hel~ in place by cotton wedged between t~e seedling " 'j, and autotro ph1c stages of seedl1ng develo p ment. The and the neck of the b?ttle. After 24 ~ours of seed~mg growth, . .' ch P and R of leaf matenal \vere determmed. Just prior to deterrol: of the en.dosperm 1.11 g~rm~natIon and the. anges minations, strips of bees,vax were wedged bet\veen the seedling whIch occur In the redIstnbut10n of metabol1tes have and th bottle neck, to seal off the top from the root environbeen thoroughly studied b)' other workers, particularly mel~t. T~us, CO, exchange determinations .\,-ere made on the I .n corn (2 3) Little is known however concerning top. only, bllt as affected by the absence or presence of endo1 .'.., " sperm. Measurements of P and R were made m a leaf chamber the relative contrIbutions of endosperm and ne,v leaf at 21.8 C, and 30% relative humidity. Light energy during P material to the total energy of the plant. ,vas 0,33 cal/cm'/min pro,ided by three 500-\vatt photo flood In P revious studies on the effect of tem p erature fila~ent !ight~. Measurements of P an.d dark R were made by . . . passmg air \Vlth a CO, content of 325 ppm through the leaf stress on seedling gro~.th, rate of endosperm utIl1zachamber and measuring the increase or decrease in CO. of air tion was used as a criterion in delineating the stage coming out of the leaf chamber with an infra-red gas analyzer. of seedling de,elopment. Green shoots developed 10llO' Flo\v of air through the chamber \vas controlled at rates of from bef?re the en~osper~ '''as completely. utilized (1, .4f ~r~e;t~iters per min, depending on the amount of leaf material ThIS observauon raIsed several questions concermng At each seedling age, P and R measurements were made on energy relationships. What is the importance of endothree to sLx replications of seedlings ,vith or \\ithout endosperm, I spenn once the green shoot has emerged? Is its rate of ~llm~ers ,of seed~ings 'per replication ,vere ,~ec.reased ~it? age t'l. t'o ffected by the Presence of the green shoot and size m order to keep air flow rates \'Ithm .the lImits of u 1 1Za 1 n.a .. . Ollr flo\\.meters, and ,vere 5. 3, 3, 2, and 1. respectively for ages and what IS the contrIbutIon of the latter to the seedof 9, 1°, 11, 12, and 13 days. Follo,ving each determination. ling while energy is still available from the endospenn? leaves \vere clipped at the surface of the \vax on each bottle. traced on paper, oven-dried. and ,veighed. Rolled or partially I rolled leaves \vere not unrolled for tracings of leaf area, Leaf 1 Contribution from theCrops Research Division, Agricultural tracings were cut out and \veighed. The ratio of area to ,veight Research Service, U. S. Department of ,\griculturc and the Monof paper ,..as llscd to calculate leaf areas. tana Agricultural Experiment S~ation. Publis~ed with approval , of the director as Paper No. 98:). Journal Series, Montana Agr. 14COZ Labellmg Exp. Sta. Received July II, 1969. . . . . , , :\gronomist, Crops Research DIvIsion: -,\~S, USDA and Seedlmgs of S, 9, .'°, II. and 12 days o~ age, \vlth and lVlthout gradllate assistant, Dept. of Botany and ;\Ilcroblology, Montana cndosperm. ,vere glv~n ~'C~. to det.ermme the effe~t of endoState University. Bozeman, ~Iont. 59715. sperm removal on distrIbution of fIXed CO.. Seedlmg growth

It has been shown by Burkholder, McVeigh and Moyer,(1) by Barton-Wright,(2) and by Mather and Barton-Wright(3) that kernels of surgary maize are about twice as high in nicotinic acid as those of starchy maize. In the first-mentioned paper, kernels of a group of sugary strains were reported to average 34.6 micrograms of nicotinic acid per gram of tissue, as contrasted with 20.8 for starchy strains; in the last two papers the averages were 26.2 for sugary strains and 15.8 for starchy.

STRAUS, JACOB. (U. Oregon, Eugene.) Maize endosperm tissue grown in vitro. lII. Development of a synthetic medium. Amer. Jour. Bot. 47(8): 641-647. Illus. 1960.-The development of a synthetic medium for the growth of endosperm tissue cultures derived from the maize variety, 'Black Mexican Sweet,' is described. Previously, these tissues required yeast extract, casein hydrolyzate, or tomato juice in the medium in order to grow. The growth-supporting activity of these complexes could be attributed to their organic nitrogen content. The effect of juice extracted from fresh tomatoes is enhanced by autoclaving under acid conditions. Presumably this treatment increases the free amino acid content of the tomato juice. One-dimensional paper chromatograms of tomato juice autoclaved under acid conditions indicated the presence of a large amount of free amino acids. Addition of 1.5 X 102 M asparagine to the basal mineral-sugar-vitamin medium (White's medium plus Nitsch's trace-element solution) resulted in better growth than that supported by yeast extract, tomato juice, or casein hydrolyzate. Arginine was ineffective. Glutamine, glutamic acid and aspartic acid (all at 1.5 X 10' M) supported appreciable growth of the tissue but none of them were nearly so good as asparagine in this respect. Thus, a medium containing minerals, sugar, vitamins, and asparagine is capable of supporting excellent growth of maize endosperm tissue cultures. IN TIIE FIRST paper of this su ies (Straus and LaRue, 1954) a report was gi-ven of some of the growth requirements of corn endosperm tissue cultures. At that time, optimum growth of the tissue required, among other substances, 0.5% Seitz-filtered Difco yeast extract (YE). Preliminary experiments reported in the same paper indicated that some of the growth-promoting activity of YE could be attributed to its high organic nitrogen content. Thus, acid-hydrolyzed casein hydrolyzate (CH) or a less complex mixture of only 5 amino acids could replace about I/j of the activity of YE. LaRue (1949) and Sternheimer (1954) demonstrated that tomato juice (TJ) obtained from whole canned tomatoes was as effective in maintaining growth of the tissue as YE. However, YE was used by Straus and LaRue rather than TJ because growth supported by the latter was sometimes erratic. This was probably due to differences between the different lots of canned tomatoes. In a recent paper, Tamaoki and Ullstrup (1958) reported their work on the growth of the ends Straus, 1958) and sometimes in red tissues (Straus, 1958) . Both the colorless and red tissues have been isolated and are now maintained as separate cultures. The general methods of initial isolation of the endosperm, the general growth conditions, and

Gibberellin-like material was estimated in barley corns “malted” at 14·4° or germinated on moistened filter paper at 25°, using a lettuce hypocotyl elongation assay. α-Amylase was measured ( a ) in these samples, ( b ) in grains (endosperms) deembryonated after various malting times then incubated further, and ( c ) in endosperms dosed individually with known amounts of GA 3 (gibberellic acid), and incubated at 14.·4°. The dosing technique provides the basis for a method of detecting and estimating 0·05 mμg or less GA 3 . A relationship existed between the “gibberellin” content and the rate of α-amylase synthesis in malting grain. Time lags were detected between the appearance of “gibberellin” in the whole grain, the arrival of the “gibberellin” in the endosperm and the appearance of α-amylase. For the first time the quantitative relations between GA 3 added to individual endosperms and consequent α-amylase production were investigated. These quantitative results strongly supported the concept that “gibberellin”, originating from the embryo, is of overwhelming importance in regulating the synthesis of α-amylase in the aleurone layer in malting grain, at least when the endosperm has been hydrated and sensitized by the postulated “embryo factor”. The endogenous gibberellin behaved more like GA 3 than GA 1 , or a mixture of varying proportions. Grain germinated on moistened filter papers at 25° gave apparently anomalous results in that the maximum value of α-amylase occurred before the greatest level of “gibberellin”.

A B S T R A-C T Arceuthobium douglasii develops a dome-like structure, the ovarian papilla, in which 2 megasporocytes are formed. The papilla is not a true ovule, for no integuments are formed, and it is forced aside by the developing endosperm. Megasporocytes are differentiated in the spring, but meiosis does not occur until the following spring. A tetrasporic embryo sac is developed which is 8-nucleate at maturity. Pollination and fertilization occur approximately 13-14 months after initiation of the inflorescence. Only 1 of the 2 embryos develops after fertilization. After fertilization, the embryo sac segregates into 2 parts, one containing the zygote and the disintegrating synergids, the other the primary endosperm nucleus and the degenerating antipodals. This primary endosperm cell elongates toward the base of the ovarian papilla. Cytokinesis then forms an endosperm cell, adjacent to the zygote, and a haustorial cell. The haustorial cell forms several tiers of cells which persist during the development of the embryo and endosperm. The zygote, while still contained within the ovarian papilla, divides, forming a 2-celled sphere. It remains unchanged until after it is conveyed out of the ovarian papilla by the developing endosperm. The development of the embryo and endosperm is arrested in the autumn approximately 3 months after their initiation. They complete their development the following spring and summer. iVIOS'T previous developmental studies in the genus Arceuthobium have dealt with the endophytic system (Thoday and Johnson, 1930) and with the mature fruit. A few papers have been published which cover various aspects of megasporogenesis, embryology, and endosperm development in different species of Arceuthobium (Johnson, 1888; Thoday and Johnson, 1930; Dowding, 1931; and Cohen, 1963). Because these investigations are not entirely in agreement with one another and because some stages of embryology have not been presented, there is need for further morphological study on several species of the genus. The present paper presents only part of the ontogeny of 1 species (A. douglasii), namely, development of the megasporocyte and the embryo sac, fertilization, and early embryonic and endospermic development. MATERIALS AND METHODS-Staminate and pis

In a previous paper (7), the advantages of corn endosperm tissue cultures as an experimental tool in attacking the problem of anthocyanin synthesis were pointed out. The general culture conditions, the identity of the pigments, and the requirement for light were reported in the same paper. Thimann and Radner (11, 12) have shown that anthocyanin synthesis in Spirodela is inhibited by purine and pyrimidine antagonists, by certain sulfurcontaining compounds and certain amino acids. They also demonstrated (13) that riboflavine reverses the inhibition caused by many of these substances, and further that riboflavine substitutes for the light reaction involved in anthocyanin production by Spirodela. When work on the current problem was begun, it was expected that the effect of various chemicals on anthocyanin synthesis in both the endosperm cultures and Spirodela would prove to be similar. As this report will indicate, however, data obtained so far show that pigment synthesis in the two systems is affected differently by certain of the same compounds tested.

The occasional appearance of a maize seed, the endosperm of which is in part colored and in part colorless or in part starchy and in part sugary, has long been known, and much speculation has been indulged in by geneticists in attempts to account for the phenomenon. Some years ago the writer (Emerson, I9I5) reviewed the hypotheses that had been previously offered as possible explanations of such seeds and suggested the further hypothesis of somatic mutation, a suggestion that has been repeated, apparently independently, by J. L. Collins (I919). It was noted also that irregular chromosome behavior might possibly be concerned. In a later paper (Emerson, I9I8) numerous cases of anomalous endosperm development were reported and discussed in relation to the hypotheses of somatic mutation and of aberrant chromosome behavior. It was pointed out that the facts then at hand could be accounted for equally well by either of the two hypotheses, and the kind of evidence necessary for a crucial comparison of the two was noted. In the latter paper evidence was presented that tended to prove that aberrant seeds are not produced (i) when the dominant endosperm factor concerned, for example the aleurone-color factor C, is homozygous and therefore triplex, C C C, or (2) when the dominant factor is brought into the cross by the female parent and its recessive allelomorph by the male parent, C C c, but only (3) when the dominant factor is contributed by the male alone, c c C. In the case of either C C C or C C c, a single mutation from the dominant to its recessive allelomorph could result only in C C c or C c c, respectively, and the aleurone would still be colored and no apparent anomaly would result. To produce colorless aleurone, c c c, two mutations in case of C C c and three in case of C C C must occur simultaneously or successively in the endosperm of the same seed-a chance so small that it might well be disregarded. It was noted that a single dominant mutation from c to C should change colorless, c c c, to colored, C c c, aleurone, but the relative infrequency of dominant mutations was thought to account for the lack of observed aberrant seeds in homozygous colorless types. Similarly, it was noted that if a single non-disjunction of the chromosome carrying C or c occurred, it could not result in a visible change in aleurone color in case of such genotypes as C C C, C C c, or c c c, but only

The present paper is the second contribution to the embryology of genusPhoenix L. The development of inflorescences, anthers, ovules, female gametophyte, endosperm and embryo was studied and the results obtained are given in this paper. Previous literature and methods followed have been given in the paper dealing with the embryology ofP. sylvestris (Proc. Ind. Acad. Sci., 1968,67 B, 77–96) and hence are not repeated here. The embryo-sac is of the monosporic octonucleatePolygonum type and embryo development follows theGeum variation of the Asterad type. The embryology in these three species thus broadly agrees with that inP. sylvestris.

THERE HAS BEEN no published study of ovule development of an aneuploid apple. The degree of fruitfulness and meiotic behavior of an aneuploid is of interest to horticulturists, cytologists, and plant breeders in view of the probable allopolyploid origin of the species and the frequency of aneuploid seedlings in progeny of triploid varieties. This paper presents the results of a study of the reproductive phases of ovule development in the aneuploid apple selection New Jersey 4. The New Jersey 4 selection is an aneuploid seedling resulting from a cross of the triploid varieties Red Gravenstein X Close. There have been numerous studies (Shoemaker, 1926; Rybin, 1926; Howlett, 1931, 1932, 1938; Elssmann and von Veh, 1931; etc.) of some phase of development of the megaspore, female gametophyte, embryo and endosperm in apple but relativety few (Wanscher, 1939; Hough, 1947) have made a study of all phases and their interrelation. Maheshwari (1948) has thoroughly reviewed the literature oni development of the angiosperm embryo sac. MATERIALS AND METHODS.-New Jersey 4 was selected for study because of its many desirable horticultural and commercial characteristics, its unusual parentage and its failure to set consistently commercial crops of fruit in spite of profuse annual flowering. New Jersey 4 has a chromosome number of 44 ? 1. The diploid variety Jonathan, with 34 chromosomes, was selected to compare development of the megaspore, and female gametophyte, and of fertilization and development of the embryo and endo. sperm with similar developments in New Jersey 4. Daily collections were made from April 9 when the buds were in the "cluster bud" or "pre-pink" stage until 12 days after pollination. Periodic collections of New Jersey 4 fruit were made for 15 additional days. A crystal violet staining schedule similar to that described by Hancock (1942) was used. In this paper the descriptions given apply to both varieties studied uniless otherwise stated. DATA AND OBSERVATIONS.-A megaspore mother cell had differentiated in a small percentage of the ovules by April 7 when the flower buds were in the "cluster bud" or "pre-pink" stage of development. Typical prophase development followed early prophase, which persisted for some time. This was

In a study2 having to do with methods of hastening the germination of common asparagus seeds, it became necessary to determine the stages in the development of certain seed structures. This investigation was later extended, and the results are here presented. The seed of common asparagus arises from an anatropous ovule, two ovules normally being borne in each of the three locules of the ovary. The seed coat is black, finely rugose, and somewhat brittle. The embryo is a slender, threadlike structure completely imbedded in the hard, horny endosperm, the walls of which are hemicellulose. The structure of the mature seed of asparagus is briefly described and figured by HARZ. His descriptions and figures, however, show certain errors, which undoubtedly were due to his failure to follow through the changes which the seed structures undergo in their development. For example, he regards the structureless membrane just beneath the seed coat as the "epidermis of nucellus," whereas developmental studies show this to be of integumentary origin. Both paraffin sections and sections of fresh seeds were employed. The latter were found to be particularly helpful. All drawings were made with a camera lucida. Integument At the time of pollination, the inner integument of the ovule consists of two cell layers, and the outer integument of from five to ten layers (figs. I-3) . The line of demarcation between the integuments I Contribution from the Division of Botany, College of Agriculture, University of California. 2 BORTHWICK, H. A., Factors influencing the rate of germination of the seed of Asparagus officinalis. Calif. Agric. Exp. Sta., Tech. Paper i8. I925. 3 HARZ, C. D., Landwirtschaftliche Samenkunde. i885. 4 In all the figures of this paper the following indicators are used: de, disintegrating endosperm cells; e, endosperm; em, embryo; gr, stem growing point; I, hilum; ii, inner integument; m, micropyle; n, nucellus; oi, outer integument; pe, pitted, thick walled endosperm; pm, pectic membrane; r, root tip; rc, root cap; s, seed coat; sac, embryo sac; sm, suberized membrane; su, suspensor; te, thin walled endosperm cells. Botanical Gazette, vol. 8o] [426

A number of papers have appeared during recent )Tears on the structure and development of the embryo-sac in the fanfily Amarantacm, but not much attention has been paid to the development of the embryo. Naithani (1933) and Joshi and Rao (1934) published a few observations on D~_~era arvensis, but these are very meagre and give no adequate idea of the subject. Woodcock (1931) has described the development of the seed in Amarantus caudatus, but he deals chiefly with endosperm and perisperm. The present paper therefore is the first detailed account of embryogeny for the family Amarantace~. The plants studied include Alternanthera sessilis R. Br. and Digera arvensis Forsk., some phases of the !ire-history of which have already been described by Naithani (1933), Joshi and Rao (1934), Puff and Singh (1935), Kajale (1935) and Joshi (1936). During the present investigation, the first plant has been studied in more detail than the second, but the two have been found to agree in all essential points except in the structure of the endosperm.

IN THE PRECEDING PAPERS of this series (Buell, 1952a,b) the seed development of Dianthus chinensis was described and variations in starch deposits in ovary and ovule, fruit and seed were correlated with the periods of ovule and embryo growth. It then seemed desirable to determine whether functional relationships might be indicated by morphological changes if the usual growth conditions were varied. Brink and Cooper (1940, 1944, 1947) have observed that in normal seed development which results from the balanced growth of embryo, endosperm and maternal tissues of the ovule, the endosperm is especially important. Since varying the genetic composition of the endosperm is a convenient method of altering the circumstances of its growth, interspecific crosses offer an opportunity to test the relationships among embryo, endosperm, and ovule tissues in the seed development of Dianthus. Of the species available for crossing, the more frequent blooming of D. chinensis as compared with D. plumarius resulted in a greater quantity of material for observation with this species as the female parent. The young fruits and seeds were prepared for study as described in the first paper of this series (Buell, 1952a). According to Rohweder (1934) the species of Dianthus form a polyploid series with a basic chromosome number of 15. The number of chromosomes listed for D. chinensis varies in the reports of different investigators: according to Ishii (1930) the haploid number in the species is 15; according to D)arlington and Ammal (1945), and Rohweder (1934), after Blackburn and Gairdner, it is 30. Rohweder (1934) found that different strains of D. plumarius have haploid chromosome numbers of 45 and 15. From mitotic figures of young ovules and embryos observed in the present investigation, the diploid chromosome number appears to be 60 in D. chinensis, and 90 in D. plum

Since Galston and McCune (1961) reported that GA altered the isoperoxidase pattern of a dwarf genotype of maize, Galston and his co-workers have shown repressive and inductive effects of auxin on isoperoxidases of green pea stems and tobacco pith cells, respectively (Ockerse et al., 1966; Galston et al., 1968). In previous papers (Momotani and Kato, 1966; 1967), we have reported on the isozymes of the α-amylase induced by GA and helminthosporol (H-ol) in the embryo-less endosperm of barley and discussed their correspondence with those obtained by Frydenberg and Nielsen (1965). More recently, we obtained 18 isozymes of α-amylase from the same preparation by improved gel electrofocusing as developed by Awdeh et al. (1968). This paper presents the experimental results together with those of experiments in which synthesis of α-amylase was achieved in vitro.

In this seldom-cited paper, published in the same issue of the "Proceedings of the National Academy of Sciences" as Creighton and McClintock's paper on genetic and cytological crossingover, McClintock established the crossover frequencies and order of the genes C (red aleurone in the kernel), Sh (shrunken endosperm), and Wx (waxy endosperm).

Senescence at the cellular, organ, and organism levels is a poorly understood but nonetheless well established fact. Regardless of the level at which senescence is studied and regardless of the possible interactions between different organs and cells of different ages and functions, senescence is an expression of events at the cellular and subcellular levels. An ideal material, therefore, for studying senescence would seem to be a homogeneous population of nondividing cells; that is, a stationary phase cell suspension culture derived from a single cell. Less ideal materials from the standpoint of homogeneity, but more readily available, are the endosperm and cotyledons of germinating seeds. The cotyledons of the pea have been selected because they are a convenient size, they are hypogeous, they do not develop into green leaf-like structures, their cells are fixed postmitotic diploid without associated endosperm cells, and their normal life span is 2 to 3 weeks. This paper describes the influence of a factor or factors from the axis tissues on the course of senescence of excised pea cotyledons. Earlier papers (4, 5) reported the formation of phosphatase and amylase activities in the cotyledons of germinating peas and the fact that the phosphatase activity did not develop in the excised cotyledons. Also the mitochondria of the excised cotyledons lost phosphorylative function after about two days and oxygen uptake capacity after 3 days. In addition, the excised cotyledons failed to incorporate glycine-C14 into TCA precipitable proteins after 3 days. These observations were sufficient to indicate an important interaction between the axis tissues and the cotyledons of the germinating pea. Although it seemed logical to suppose that some beneficial factor from the axis tissues was moving into the cotyledons, it was necessary to consider the possibility that a dleleterious factor might be moving out of the cotyledons into the axis. The data reported here indicate that the interaction results from the transport of some factor or factors from the axis into the cotyledons during the 1st and 2nd day of germination. This factor has not been identified and no completely satisfactory replacement for it has been found.

WVirwille and Mitchell reported that applications of 2'-isopropyl-4'(trimethylammonium chloride) -5'methylphenyl piperidine-1-carboxylate (Amo 1618) (fig 1, I) to several types of flowering plants led to an abnormal growth habit characterized by shorter stems and internodes (17). Amo 1618 is one of the most active of several carbamate esters containing a quarternary ammonium group which produce this effect (12). Cathey has recently reviewed the physiological effects of these and other plant growth retardants (2). Sachs et al. (15) have shown that application of Amo 1618 leads to a drastic reduction of mitotic activity in the subapical meristem of vegetative, rooted cuttings of Chrysanthemum morifolium. In this case, as in numerous other instances reported in the literature, added gibberellins partly or completely overcome the action of plant growth retardants. [see (2) for a review]. Kende, Ninnemann and Lang (11) have provided evidence for the proposal that at least some of these plant growth retardants may function by inhibiting the production of gibberellins in the plant. This proposal was based initially on their demonstration that the addition of Amo 1618 at 100 ug per ml or of f-chloroethyltrimethylammonium chloride (CCC) at 300 ,ug per ml to the culture medium of Fusarium moniliforme Sheld. leads to a complete inhibition of gibberellin production without affecting mycelial growth (11). Several tvpes of evidence were presented to show that CCC inhibited the biosynthesis of gibberellins rather than causing destruction of preformed gibberellins (13). More recently Baldev et al. have also shown Amo 1618 to be an inhibitor of the accumulation of substances with gibberellin-like activity in developing pea seeds (1). The evidence to be discussed in this paper shows that an enzymic reaction resulting in the production of (-)-kaurene (fig 1, Ia) in a flowering plant system is inhibited in the presence of Amo 1618 or certain other plant growth retardants. (-)-Kaurene is a probable intermediate in the biosynthesis of gibberellins. Not only did Cross et al. (5, 6) isolate this compound from culture filtrates of F. noniliforme along with gibberellins, but they also demontrated in these cultures a conversion of (-) -kauretne labeled with C14 in the exocyclic methylene group to C14-gibberellic acid (fig 1, III) labeled in the same positioni (4). Recently we have confirmed this finding with C'4-labeled (-)-kaurene (9). The substrate in this case was derived from 2-C14-mevalonate in the endosperm nucellus of Echinocystis macrocarpa Greene (wild cucumber) seed. Furthermore, Phinney et al. (14) have reported that (-)-kaurene promotes growth of the dwarf-5 mutant of Zea mays, an effect qualitatively indistinguishable from that induced by an added gibberellin. This evidence considered together strongly supports the idea that (-)kaurene is an intermediate in gibberellin biosynthesis. Thus, an inhibition of (-)-kaurene synthesis would result in an inhibition of gibberellin synthesis. The endosperm of E. macrocarpa seed, a source knowln to be relatively rich in gibberellins and gibberellin-like substances (3,8,16), was used in the studies reported in this paper as a source of enzymes which catalyze the formation of (-)-kaurene. (-)kauren19-ol (fig 1, IIb) and trans-geranylgeraniol (fig 1, IV) from mevalonate (7, 9). Since these diterpenoid metabolites are involved in the gibberellin biosynthesis pathway, a study of the effects of Amo 1618 and other growth retardants on their formation was undertaken.

Stimulation of endosperm metabolism by the addition of exogenous gibberellic acid (GA) has been reported by many workers. A series of papers by Paleg (8, 9, 10) have described the dependence of loss of dry weight, starch hydrolysis and protein release in excised barlev endosperm on the presence of added GA. Work with many varieties of barley, wheat and oats has confirmed the generality of this effect (11, 12). Since GA-like compounds occur in barley embryos (5) and in many higher-plant tissues (1, 7), it is likely that endosperm metabolism is initiated by the release of such compounds from the embryo during normal generation. More detailed studies have been made on the GA stimulation of a-amylase activity in barley endosperm in an attempt to elucidate the mechanism of action. Paleg (10) has suggested that the hormone activates or releases proteases, which in turn activate or release the a-amylase; whereas MacLeod and Millar (6) proposed that GA brought about the release of hydrolytic enzymes, including a-amylase from lysosome-like particles. Recently, however, Briggs (2) has reported an enhanced de novo synthesis of proteins in the aleurone cells of barley endosperm in response to GA, and Varner (13) has demonstrated the stimulation of de novo synthesis of a-amylase in the same tissue. This paper reports the results of experiments designed to test whether carbohydrate and protein catabolism in excised corn endosperm is dependent on exogenous GA as in other cereal grains. In addition, the effects of GA on the activities of ribonuclease and 3'nucleotidase in the endosperm were examined. These 2 enzymes are invo!ved in nucleic acid catabolism, and their activities increased markedly during germination (4).

The present experiments were conducted with the object of determining the cause for varietal difference in speed of seed germination. The experiments performed in connection with this study were grouped into two parts, namely (a) the relation between germination and glycolysis in rice seed and (b) the type of germination. The results obtained are summarized as follows. (1) the differential speed of germination of each variety has the same tendency as the strength of glycolysis in endosperm and amylase activity in embryo and endosperm. (2) the type of germination seems to be influenced by the presence of endosperm, that is, at the high temperature (30°C) the radicle appears previous to the plumule while at the low temperature (15°C) this sprouts more quickly than that on filter paper. But on the germination of the embryo cut off from the endosperm, regardless the temperature, the plumule appears previous to the radicle. The term "germination" is used in this paper to mean the phenomenon in which the plumule or the radicle appears when the rice seed is placed on filter paper or in water.

Paper-chromatographic analysis was done on the sugar contained in embryo and endosperm of the lowland rice seed, and of each of the youngest seedling and its endosperm, i. e., Norin No. 41 (translucent grain) which requires more oxygen for germination and Hikotaro-mochi(non-translucent grain) which requires less, both being germinated at 30°C on the water blotted paper, in water 5 cm depth, and in water 5 cm depth and vacuumed by rotary pump. Sugars detected in this experiment were raffinose, sucrose, glucose, fructose and two unidentified sugar-like substances. Raffinose was disappeared as germination progressed, but galactose to be produced by its hydrolysis was not found. Sucrose was always detected in seeds regardless of the different germinating conditions. It was considered that hexoses were synthesized into sucrose as a precusor-like type or a translocating type. Maltose which should be naturally produced from starch was not fund. It may be produced from starch by amylase in vivo, without regards the weak intensity of its reaction, because pH values of cell saps in the embryo and the endosperm of two kinds of seeds are greater than optimum value in vitro. Hexose were found in each of two kinds of seeds at 4-8 hours after the imbedding on the water blotted paper or by submerging in the water of 5 cm depth, but these were found only in the endosperm during 4-20 hours in the anaerobic condition while in embryo and endosperm it takes 24 hours. From above results, as disclosed dy DASTUR and DESAI (1935), enzymic activities in the germination were pormoted by a successive supply of oxygen, while these were inhibited under the anaerobic condition, particularly invertase was concerned. In this connection, it is considered that hydrolysis of sucrose by invertase is the most important factor for the germination and the suceeding growth of the seedling.

Using seeds of three varieties both upland and lowland rice, non-volatile organic acids occurred in germinating stage under three germinating conditions on the water blotted paper, in water (Subdivided to 0.6 cm and 5 cm depth) and in vacuum were examined by paper-chromatography. 1) In the ungerminated seeds, oxalic, citric and malic acids were found in both embryo and endosperm of upland and lowland rice seeds, however, glycollic, α-ketoglutaric, lactic, succinic and fumaric acids were found with the passage of germination time. 2) From these facts, it is suppposed that the Krebs cycle occurs in germinating stage of rice seeds, both embryo and endosperm. 3) In embryo, the amount of malic acid is larger than citric acid, while citric acid is larger than malic acid in endosperm, however in embryo, in vacuum, citric acid is more abundant than malic acid before 36 hours of germination time, and at the 72 hours, for the first time, malic acid became much more than citric acid. 4. In vacuum, both sort and quantity of acids were less than in two other germinating conditions. 5. No remarkable differences were found between upland and lowland rice Seeds, and Uruchi translucent grain) and Mochi (non-translucent) types.

The changes in cytoplasmic fine structure during the interval between pollination and zygotic division in the megagametophyte of Zea mays are reported. The rate of membrane synthesis appears to be low in the mature unfertilized megagametophyte. This megagametophyte is suggested to be in a state of relative metabolic inhibition. The rate of membrane synthesis is high in the zygote and free nuclear endosperm. After fertilization the swelling of the mitochondria in all cytoplasms is interpreted to reflect a presumed increase in metabolic rate relative to growth. In the zygote and endosperm, endoplasmic reticulum (ER) production is associated with the nuclear envelope. In all of the material the ribosomes not associated with the ER are helical polysomes which increase in length after fertilization. On the basis of permanganate staining, the ribonucleoprotein of the endosperm is suggested to be different from that of the other cytoplasms. The specific plastid form of each cell type does not change during the first few hours after fertilization. Dictyosomes are most numerous and active in the antipodals. They are least numerotus in the egg and zygote. The smallest dictyosome vesicles are in the synergids. Dictyosome number and vesicle size increase in the egg and central cell after fertilization. Similarities among the megagametophytes of several angiosperms are enumerated. THE EMBRYO SAC is uniquely the megagametophytic phase in the life cycle of angiosperms. The developmental pathways, final organization of the mature megagametophyte, and the details of fertilization differ greatly among species (Vazart, 1958). Electron-microscopic studies show that this variation in structure extends also to the sub-cellular level even among identical cell types (van der Pluijm, 1964; Jensen, 1965a, b; Diboll and Larson, 1966; Vazart, 1966; Rodkiewicz and Mlikulska, 1967). Among these studies the work on Gossypium hirsutum by Jensen (1964, 1967) is unique for its inclusion of the initial stages of embryogeny and for its comparative application of methods of electron microscopy, physiology and histochemistry. As part of a continuing study of the female reproductive system of Zea mays, the present paper describes the changes in the cytoplasmic fine structure during the interval between pollination and zygotic division (ca. 30 hr). Zea mays was selected because of the large amount of information already available on the species and for the ease with which the material can be handled. A comparison of the data introduced in this paper with those from the above studies should contribute to a better understanding of the angiosperm megagametophyte, structural 1 Received for publication 4 January 1968. Supported by U.S. Public Health Grant No. 1 ROI GM 13517. The author expresses his appreciation to Miss Dagmara Davis for technical assistance. 2 Present address: Department of Biology, Macon Junior College, Macon, Ga. changes relative to fertilization in angiosperms, and to the total knowledge of the corn plant. MATERIALS AND METHODS-Individual plants of commercially available 'Golden Bantam' corn were grown during the summer in 2-gal plastic containers in the greenhouse. The planting mix consisted of soil, peat and vermiculite. The plants were watered daily with a weak Hoagland's solution supplemented weekly with Bandini Gro-Rite commercial fertilizer. Day length was extended to 2:30 AM with 300-w incandescent bulbs producing 25 ft-c of light at bench level. This latter procedure was adopted to satisfy the requirements of continuing other species in a vegetative state. Temperature was maintained above 70 F at all times. Corn ears were bagged before emergence of silk. Unpollinated ears were collected 1 to 2 days after silk emergence. Fertilized material was collected 22, 24, 28, and 32 hr after hand pollination. Approximately 1-mm-thick sagittal sections of ovules containing the megagametophytes were cut from the ear and placed immediately in cold 3% or 6% glutaraldehyde buffered to pH 7.0 with 0.1 M sodium cacodylate. Megagametophytes surrounded by nucellar tissue (megasporangium) were excised from these sections while immersed in the fixative. Following 6 hr of fixation, the tissue blocks were washed in distilled water or buffer; post-fixed in either 5% unbuffered KMnO4 for 12 hr or in buffered 1 % OS04 for 12 hr; washed again, and then stained for 24 hr with 5 % aqueous uranyl acetate. The

The mechanism of seed dormancy at low temperatures (15-9°C) was investigated in the seeds of Syringa josikaea, S. reflexa and S. vulgaris. Low temperature dormancy in Syringa species was mainly imposed by endosperm embedding the radicle. Different degrees of embryo dormancy may occur in S. reflexa seeds. In most cases the low temperature dormancy was broken completely by removing the endosperm around the radicle. The endosperm did not seem to contain significant quantities of germination inhibitors, and the results indicate that it prevents germination mainly due to its mechanical resistance. The mechanical resistance of endosperm did not change during chilling or during induction of dormancy by high temperature incubation. The strength of the endosperm decreased rapidly in non-dormant seeds before visible germination. Similar changes were not observed in dormant seeds. Generally, the strength of the endosperm was lower in the non- (or less) dormant species S. josikaea and S. vulgaris than in the more dormant S. reflexa seeds. The growth potential of the embryos, measured as their ability to germinate in osmotic solutions (mannitol or polyethylen glycol 4000), was increased by chilling and by GA3-treatment. The growth potential of untreated S. josikaea and S. vulgaris embryos was generally higher than that of S. reflexa embryos. Acid ethyl acetate fractions of methanol extracts from embryos of all three species contained substances with GA3-like activity in the lettuce hypocotyl test. The activity was found at Rf 0.9–1.0 on paper chromatograms run in distilled water. No significant changes in the activity were detected during chilling or prior to visible germination.

INCREASING ATTENTION is being paid to the extraction of auxin2 from the tissues of higher plants, particularly as regards reliability and yield in relation to various extraction procedures. A comparative study of eight different methods of auxin extraction from maize endosperm has been reported (Avery, Creighton and Shalucha, 1940), the methods dealt with being those formulated in the preceding five years by several different workers. From the standpoint of auxin yield per unit weight of tissue, two of the methods were outstanding: (1) successive extractions of the same batch of tissue with several different solvents (alcohol-chloroform-water), referred to as a multisolvent method; (2) extraction with water alone as a solvent. Our object at this time is to report further studies on these two methods for hormone extraction of maize endosperm. Yields have been increased to twenty times those previously reported from this laboratory. MATERIALS AND METHODS.-For reasons pointed out in the previous report, endosperms of maize (Zea mays L. var. Canada Flint3) are again used for auxin extraction studies. Skoog's (1937) "deseeded" Avena test method was used for auxin assays, the test schedule used already having appeared in publications of this laboratory. Agar block size (10 cmm.), units used, etc., are discussed in detail by Avery, Creighton and Shalucha (1941). The Avena test laboratory was maintained at 250C., 85-90 per cent relative humidity, and lighted only with phototropically inactive light. All yields4 are expressed on the basis of air-dry endosperm (ca. 9 per cent moisture) in terms of TDC/gm. The TDC unit has been used through1 Received for publication March 1, 1941. 2 In this paper, any compound which causes curvatures away from the applied agar block in the Avena test, is considered an "auxin." 3 Courtesy of Dr. Ralph Singleton, Connecticut Agricultural Experiment Station. 4 TDC=total degrees curvature in the "deseeded" Avena test method, and is calculated from the expression CX lbOX V w

DNYANSAGAR, VISHNU R., AND DELMER C. COOPER. (U. Wisconsin, Madison.) Development of the seed of Solanum phureja. Amer. Jour. Bot. 47(3): 176-186. Illus. 1960.-Ontogeny of the seed of Solanum phureja Juz. et Buk. is described. The megagametophyte, during the course of its development, ruptures the nucellus and at maturity lies in direct contact with the inner layer (endothelium) of the single massive integument. The mature megagametophyte, a 7-celled structure, consists of a 3-celled egg apparatus, an endosperm mother cell with fused polar nuclei and 3 persistent antipodals. Both 2and 3-celled mature pollen grains are formed within anthers of the same flower; hence this character cannot be considered of any taxonomic value. Double fertilization occurs between 24 and 72 hr. after pollination. A cellular endosperm is formed, the peripheral layer acting as an absorbing tissue during the early ontogeny of the seed. Later this layer becomes organized as an aleurone layer and thereafter the source of nutrients is via the basal portion of the endosperm immediately adjacent to the apical end of the vascular tissue of the developing seed. Embryo development follows the Nicotiana variation of the Solanad type. The mature testa is composed of an outer layer of thick-walled epidermal cells, an inner layer of thin-walled cells and an intervening mass of disorganized tissue. In those instances where the ovule or young seed aborts, the endothelial cells of the integuiment become hyperactive and proliferate to such an extent that the space formerly occupied by the gametophyte or the developing endosperm and embryo becomes completely filled with endothelial tissue. THE FAMILY Solanaceae contains some 2,000 species, three fourths of which belong to the genus Solanwn (Benson, 1957). Detailed analyses of the developiment of either the seed or the embryo in this largest genus are sparse and almost negligible in the Section Tuberarium, which is economically important (Schnarf, 1931; Johansen, 1950; Beamish, 1955; Walker, 1955; Williams, 1955; Wangenheim, 1957; and Lee and Cooper, 1958). None of the investigations on the Section Tuberarium gives a detailed account of the endosperm development and particularly of the embryogeny with respect to aspects as the architecture of embryo, destination of proembryonal cells and furidamental regions of the embryonic body, and laws of embryonic development. Several wild and cultivated diploid tuber-bearing species of Solanum possess desirable features not found in the common potato, S. tuberosum. S. phureja (2n -24) is one which is of interest to potato breeders, since it matures early, possesses general virus resistance and is immune to the "Y" virus. A detailed study of normal development of seed in such a species may prove helpful in methods of overcoming difficulties of hybridization. It m-ay also be of value in furnishing a basis for determining various genetic and environmental influences on the endosperm and embryo development and in embryo-culture. 1Received for publication June 27, 1959. Paper No. 755 from the Department of Genetics, University of Wisconsin, Madison. 2 The first author expresses his gratitude to Dr. R. W. Hougas for suggestions and facilities at the greenhouse. Thanks are also due to the Ministry of Education, Government of India for award of a scholarship. MATERIALS AND METHODS.-S. phureja (Hawkes, 1956 ) (S. rybinii of U.S.D.A. Plant Int. P. I. 225682.2) and S. phureja (P. I. 205561.1) were used as the seed and pollen parents respectively. The plants were grown in the greenhouse at a temperature of 70?F. Daily collections of ovaries or developing fruits were made for 30 days starting 24 hr. after pollination. The material was fixed either in formalin-acetic-alcohol or Randolph's modification of Navashin's fluid (Johansen, 1940). Carpels for the study of pollen tubes were collected for 4 days following pollination and fixed in the latter fixative. After embedding in paraffin, sections were cut at 10-16 ju and stained with either Harris' haematoxylin, Delafield's haematoxylin, or with safranin and fast green. Styles dissected for study of pollen tubes were stained with either acetoorcein or acetocarmine. OBSERVATIONS.-Ovule and inegagametophyte.Ovules are either anatropous or campylotropous and attain the maximum length of 0.15 mm. at the time of fertilization. The nucellus is usually completely utilized during the course of development of the megagametophyte, so that the latter lies in direct contact with the innermost layer (endothelium) of the single massive integument (fig. 5). Occasionally, remnants of the nucellar tissue persist in some ovules. The endothelium consists of radially elongated cells with dense cytoplasm and prominent nuclei and persists as a tapetum-like, nutritive layer adjacent to the embryo-sac. Some of the cells of the funiculus at the chalazal end of the ovule elongate and differentiate into a vascular strand thus establishing a connection with the vascular tissue of the placenta. The megagametophyte (fig. 6) consists of 7

The presence of multiple forms of alpha-amylase in gibberellic acid-treated embryoless barley half-seeds was demonstrated by separation on diethylaminoethyl-Sephadex and isoelectric focusing polyacrylamide gel disc electrophoresis. Two major alpha-amylase fractions (A and B), each consisting of two to three isozyme components, were purified. alpha-Amylase fractions A and B were distinguishable in their reaction patterns. The optimal pH of fraction A alpha-amylase was found to reside in the acidic side (pH 5.0), as was determined by analyzing the reducing sugars formed as well as the paper chromatographic detection of reaction products. At neutral pH, 6.9, fraction A exhibited weak amylolytic activity in forming maltose. The alpha-amylase activity in fraction A was markedly stimulated by heat treatment (70 C/15 minutes). Fraction B, constituting a major part of amylases in the endosperm extract, was also found to be composed of alpha-amylase, as evidenced by the loss of enzyme activity upon allowing fractions A and B to stand at pH 3.3 for a prolonged period. The possible physiological function of the two different types of alpha-amylase in the carbohydrate breakdown of barley seeds is discussed.

Caryopses of a dormant strain of Avena fatua L. fail to germinate on moist filter paper when immature whereas non-dormant caryopses germinate at 10 days after fertilization and at later stages of development. Embryos of the dormant strain excised at any stage of development fail to germinate in a complex nutrient medium which supports germination and growth of non-dormant embryos at, and after, 10 days following fertilization. Gibberellicacid (GA) promotes germination of dormant caryopses and excised dormant embryos before and at maturity in the presence of either endosperm or the nutrient medium. This requirement for gibberellin is not absolute, since germination of dormant embryos in mid-development occurs when the nutrient medium is solidified with agar in the absence of gibberellin. The stimulatory effect of solidifying the medium is probably due to differential leaching action upon embryos affecting levels of endogenous gibberellins. The genetic source of endosperm does not influence germinability of the embryo, and endosperms of both dormant and non-dormant strains show an increasing capability of supporting postgerminal growth of non-dormant embryos. A block to the use of these nutrients by the embryo, reversible by gibberellin, is present at maturity in endosperm of the dormant strain.

It has long been known that the grains of the Darnel contain a poisonous body (Lolium), which can be extracted by ether. This substance has marked toxic action on rabbits and certain carnivorous animals, and is said to induce vomiting and other unpleasant symptoms in man, but to affect pigs, cattle, and geese but little, or not at all. In 18981 attention was drawn to the fact that a large percentage (80-100 per cent.) of the grains of this grass contain a definite fungus-mycelium, always situated in a definite layer of the seed,i. e., in the remains of the nucellus, just outside the aleurone-layer of the endosperm.

Heterosis, the evident superiority of a hybrid to its parents, has not been defined in precise genetic terms. The reasons given for heterosis are for the most part rather general and vague. A recent statement is made that heterosis is the phenotypic result of gene interaction and occurs because the heterozygote either masks different detrimental recessive genes which are homozygous in the parents, or is adaptively superior to both parents.' About 30 years ago, East2 suggested that "physiologically active" genes control speed of reactions responsible for heterotic expression. Admittedly, the elucidation of the genetic mechanism(s) responsible for heterotic expression is not as yet possible. Much work needs to be done to describe precisely the required genetic constitution of heterotic individuals, especially when these are compared to the nonheterotic ones. And the time is ripe for such work to be done. It is obvious that an alternative approach to the understanding of heterosis would be through studies of mechanisms or reactions responsible for growth and development in general, since heterosis, for the most part, is a manifestation of a superior growth rate of a hybrid over its parents.3-6 Information on the biochemistry and physiology of heterosis is scant mainly because much of the effort has been directed toward the elucidation of the genetic constitution required for heterotic expression. Cherry and co-workers have shown, however, that a maize hybrid generally had higher quantity of embryonic axis nucleotides than did the inbreds and that RNA was synthesized at a much faster rate in the hybrid, and suggested that hybrid vigor involved the plant as a whole and that reaction speeds were intimately involved.7 However, the study of the reactions per se would not be sufficient. Rather, the agents controlling these reactions should be elucidated. It is felt that then we would be taking a big step in the direction of the answer to the question, "How is heterosis accomplished?" The present paper is one of a series of reports8-13 intended to elucidate a physiological-biochemical basis for heterosis. It is concerned with early postgermination physiological changes occurring in the endosperm and seedlings of hybrid corn. Observations showing differences in respiration, liberation of reducing sugars, and amylase activity are presented in an attempt to shed light on the mechanism of hybrid superiority in terms of growth. A possible means of control of this mechanism is suggested. Materials and Methods.-Germinating seeds and seedlings of several maize single crosses and their parents were used throughout this study. The strains used were the dent inbreds Ohio 43, Ohio 45, West Virginia 5, and their single crosses W. Va. 5/Ohio 43, and Ohio 43/Ohio 45 (supplied by M. W. Johnson, West Virginia University). 212

The de novo synthesis of α-amylase in barley endosperm and isolated aleurone layers is induced by 3′,5′-cyclic purine mononucleotides and gibberellic acid. The induction of α-amylase by cyclic purine mononucleotides is prevented by 2,4-DNP, inhibitors of RNA and protein syntheses, CCC, AMO-1618 and phosfon. The induction of α-amylase formation by 3′,5′-cyclic purine mononucleotides, but not by gibberellic acid, is also blocked by inhibitors of DNA synthesis. Extracts from cyclic AMP-treated endosperm halves exhibit a characteristic gibberellin-like activity which is detectable within 12 hours from the addition of the cyclic AMP. On paper chromatograms this gibberellin-like activity is located at the Rf typical for GA3. Its formation is prevented by inhibitors of DNA synthesis, CCC and AMO-1618. Glucose inhibits the formation of α-amylase induced by gibberellic acid. Glucose has no effect on the cAMP-induced gibberellin biosynthesis. The evidence shows that the cyclic purine mononucleotides induce DNA synthesis, which results in gibberellin biosynthesis, which in turn activates the synthesis of α-amylase.