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.     

GROWTH OF THE EMBRYO OF GINKGO BILOBA UNDER EXPERIMENTAL CONDITIONS. III. GROWTH RATES OF ROOT AND SHOOT UPON MEDIA ABSORBED THROUGH THE COTYLEDONS

Ball , Ernest . (North Carolina State Coll., Raleigh.) Growth of the embryo of Ginkgo biloba under experimental conditions. III. Growth rates of root and shoot upon media absorbed through the cotyledons. Amer. Jour. Bot. 46(2) : 130-139. Illus. 1959.—Mature embryos of Ginkgo biloba were grown by inserting the cotyledons into agar medium containing one of the naturally-occurring sugars of the seed. These sugars were utilized at 1, 2, 4, 8 or 16% (w/v). Root growth was best on the sugar-mineral-water media and occurred at its maximum in media containing 4% sugar (ca. 0.25 M). The sugars may be listed as follows in order of decreasing effectiveness in root growth: glucose, sucrose, levulose, raffinose, galactose. Since very different growth rates of roots occurred on media of the same osmotic values when the latter were determined by the various sugars, it is suggested that the root utilized the entire sugar molecule as a unit whether it was mono-, di-, or trisaccharide. Such media were not effective in supporting shoot growth. Outstanding growth of shoots was obtained when either glutamine or coconut milk was placed in the culture medium. It is concluded that root growth can occur at a substantial rate by utilizing sugar and manufacturing organic nitrogen from nitrates. Shoot growth, in contrast, appears to require sources of organic nitrogen such as may be furnished by glutamine or coconut milk. These phenomena are thought to constitute fundamental differences between the metabolism of root and shoot of this embryo. Mannitol caused severe inhibition of growth of both root and shoot. It is probably toxic to this embryo. Indoleacetic acid caused severe inhibitions of growth of both root and shoot at all except the lowest concentrations utilized.     

Study of aberrant morphologies and lack of conversion of somatic embryos of chickpea (Cicer arietinum L.)

Summary Somatic embryos which originated from mature embryo axes of the chickpea (Cicer arietinum L.) showed varied morphologies. Embryos were classified based on shape of the embryo and number of cotyledons. “Normal” (zygotic-like) embryos were bipolar structures with two cotyledons and a well-developed shoot and root apical meristem, whereas “aberrant” embryos were horn-shaped, had single and multiple cotyledons, and were fasciated. Histological examination revealed the absence of a shoot apical meristem in horn-shaped embryos. Fasciated embryos showed diaxial fusion of two embryos. Secondary embryogenesis was also observed, in which the embryos emerged from the hypocotyl and cotyledonary region of the primary somatic embryo. This report documents the absence of an apical meristem as a vital factor in the lack of conversion of aberrant somatic embryos.     

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.     

LIGHT-MEDIATED INHIBITION OF IN VITRO DEVELOPMENT OF RUDIMENTARY EMBRYOS OF ILEX OPACA

The mature seeds of Ilex opaca Ait. contain rudimentary heart-shaped embryos. When excised embryos of cv. Farage were grown in vitro with a 16-hr photoperiod, only 20 % reached germination size after 13-day incubation, while 75% of the embryos reached the same stage when incubated in darkness. This light inhibitory effect increased with the length of daily light exposure as the daily photoperiod reached 4 hr. Nearly 50% growth reduction resulted as the cultures were preincubated in light continuously for 2 days before a 10-day dark incubation. After 4 days of light incubation the inhibitory effect could no longer be reversed by the subsequent dark incubation. Once the heart-shaped embryos started to grow in darkness they become progressively insensitive to subsequent light inhibition. After a 6-day initial dark incubation the embryos become immune from the inhibitory effect of light. This light inhibitory effect on in vitro embryo growth was universal in all 11 tested cultivars.     

Dark pretreatment improves adventitious shoot organogenesis from cotyledons of diploid watermelon

The influence of light incubation during embryo germination on shoot organogenesis from cotyledons of four diploid watermelon [Citrullus lanatus (Thumb.) Matsum. & Nakai cultivars was examined. Germinating embryos in darkness significantly improved the number of explants that produced harvestable shoots during the 6 week incubation period on shoot regeneration medium under a 16-h photoperiod. The percentage of explants with shoots more than doubled for `Crimson Sweet' and was about 1.5-fold greater for `Sweet Gem' and `Yellow Doll' when embryos were germinated in darkness. The percentage of explants with shoots was not significantly improved for `Minilee' by pretreating seedlings in darkness. This study demonstrates that optimal shoot regeneration can be obtained by germinating embryos in darkness before preparing cotyledon explants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of 2,4-dichlorophenoxyacetic acid (2,4-D) in somatic embryogenesis on cultured zygotic embryos of Arabidopsis: cell expansion, cell cycling, and morphogenesis during continuous exposure of embryos to 2,4-D

The relationship between cell expansion and cell cycling during somatic embryogenesis was studied in cultured bent-cotyledon-stage zygotic embryos of a transgenic stock of Arabidopsis thaliana harboring a cyclin 1 At:β-glucuronidase (GUS) reporter gene construct. In embryos cultured in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), following a brief period of growth by cell expansion, divisions were initiated in the procambial cells facing the adaxial side at the base of the cotyledons. Cell division activity later spread to almost the entire length of the cotyledons to form a callus on which globular and heart-shaped embryos appeared in about 10 d after culture. Anatomical and morphogenetic changes observed in cultured embryos were correlated with patterns of cell cycling by histochemical detection of GUS-expressing cells. Although early-stage somatic embryos did not develop further during their continued growth in the auxin-containing medium, maturation of embryos ensued upon their transfer to an auxin-free medium. In a small number of cultured zygotic embryos the shoot apical meristem was found to differentiate a leaf, a green tubular structure, or a somatic embryo. Contrary to the results from previous investigations, which have assigned a major role for the shoot apical meristem and cells in the axils of cotyledons in the development of somatic embryos on cultured zygotic embryos of A. thaliana, the present work shows that somatic embryos originate almost exclusively on the callus formed on the cotyledons. Other observations such as the induction of somatic embryos on cultured cotyledons and the inability of the embryo axis (consisting of the root, hypocotyl, and shoot apical meristem without the cotyledons) to form somatic embryos, reaffirm the important role of the cotyledons in somatic embryogenesis in this plant.     

MORPHOGENETIC ASPECTS OF A LEAFLESS MUTANT IN TOMATO I. GENERAL PATTERNS IN DEVELOPMENT

The embryo of the reduced form of the lanceolate mutant in tomato fails to undergo the heart-shaped stage of development. Cells in the shoot apical region of this leafless mutant lose their meristematic character and develop into mature parenchyma during embryogenesis. This early loss of meristem tissue leads to the determinate growth which is evident in the seedling. In contrast to normal, starch grains are visible with the light microscope in cells of the shoot tip of the mutant hypocotyl from early embryogeny up to and including the seedling stage, and protein bodies are abundant in the same tissue of fully developed mutant embryos. The shoot apical region in homozygous mutant embryos with cotyledons or cotyledon-like structures exhibits some cytochemical and morphological similarity with the normal shoot apex. Morphological variation in these forms appears to be in a continuous pattern. The extent of their development and consequent longevity is related to possible differences in rates of cell expansion and variation in environmental factors during the early stages of embryogenesis.     

SOME ASPECTS OF MORPHOGENESIS IN CUSCUTA GRONOVII

More than 100 primary root segments from dormant seeds, 100 radicular segments from immature embryos, 450 whole immature embryos, and a number of whole seedlings of C. gronovii were studied under in vitro cultivation in the presence of such growth-regulating factors as: coconut milk, casein hydrolysate, auxins, kinetin, adenine, gibberellic acid, and additional B vitamins as supplements to White's medium. In normal embryogeny early swelling of the basal pole of the embryo and profuse production of epidermal hairs upon germination are superficial characteristics of Cuscuta which resemble seedling root growth in other plants. At no time is there evidence of a primary-root meristem, nor at any time do cotyledons develop. It was not possible to induce root formation experimentally from any of the starting embryonic materials, nor from stems which were cultured to maturity culminating in the production of flowers after 6 months of in vitro cultivation, nor from callus derived from the radicular pole of the embryo. The undifferentiated radicular pole frequently gave rise to rapidly growing callus from which new shoots were formed spontaneously, although some immature embryos completed normal embryogeny. Embryo size and subsequent type of development were not strictly correlated but there was a tendency toward the following patterns. Embryos less than 0.4 mm in length lost their original form, callused evenly throughout and became masked by development of buds over the entire surface. Embryos 0.4–1.2 mm were most apt to retain their original form; callus developed at the radicular pole but not at the shoot pole. Embryos 1.5 mm and larger frequently gave rise to radicular callus but also grew normal shoots. Embryo inoculants which had begun to coil were most apt to complete normal embryogenesis. No correlation could be drawn between the addition of specific growth regulators or combinations of regulators and specific patterns of development. It is suggested that in the evolution of Cuscuta from autotrophic ancestors, the loss of both cotyledons and roots was a single embryonic event and so complete that even under the influence of a highly modified biochemical environment it would be impossible to derive roots from dodder tissue. The usual interpretation of the dodder haustorium as a highly modified adventitious root in all probability is not valid. Is is further suggested, however, that this loss of root and cotyledon is not a necessary consequence of the evolution of the parasitic mode of nutrition, nor of the gross adult growth form.     

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.     

Dormancy in Seeds of Charlock: I. DEVELOPMENTAL ANATOMY OF THE SEED

The anatomy of the embryo, endosperm, and testa throughout thedevelopment of charlock seeds is described. In mature seedsthe embryos are morphologically fully differentiated. Each embryoconsists of a shoot meristem with two large cotyledons but noleaf primordia, and a root meristem with a root cap. The embryois surrounded by a single layer of aleurone-like cells derivedfrom the endosperm and enclosed within the testa. Mucilagesand phenols which could greatly retard the diffusion of oxygeninto the tissues of the embryo are found in the testa. Dormancy,which appears to be induced by a shortage of oxygen, is probablyassociated with the presence of these covering layers.     

ONTOGENY OF THE SHOOT APEX OF SEEDLINGS OF PINUS PONDEROSA

Dormant seeds of ponderosa pine (Pinus ponderosa) were stratified and then planted in the greenhouse. Changes occurring in the shoot apex during germination and growth of the seedlings were observed and are described. Cell divisions in germinating embryos are first noted on the flanks of the apex near the cotyledons. From these loci, mitotic reactivation proceeds up the flanks of the apex and inward toward cells of the rib meristem. Cells are dividing actively in all regions of the shoot apex of seedlings 12 days after planting, but no cytohistological zonation is evident due to differences in staining intensity and in cell and nuclear size. In 64-day-old seedlings the mitotic rate is reduced and the characteristic zonation of pine apices begins to appear. Zonation is even more evident in older seedlings. These observations are discussed in relation to the concept of initials and to theories concerning factors which regulate growth in the shoot apex.     

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.     

Applications of DL-buthionine-[S,R]-sulfoximine deplete cellular glutathione and improve white spruce (Picea glauca) somatic embryo development

In white spruce (Picea glauca), an improvement of somatic embryo yield and quality can be achieved by applications of dl-buthionine-[S,R]-sulfoximine (BSO), which inhibits the biosynthesis of reduced glutathione (GSH), thereby switching the total glutathione pool towards its oxidized form (GSSG). Applications of BSO almost tripled the embryogenic output of two cell lines by increasing the number of embryos produced by 100 mg⁻¹ tissue from 65 to 154 in the (E)WS1 line and from 59 to 130 in the (E)WS2 line. This increase in embryo number was ascribed to a higher production of morphologically normal embryos with four or more cotyledons (group A embryos), at the expense of group B embryos, characterized by fewer cotyledons. The quality of the embryos produced, estimated by their post-embryonic performance, was also different between treatments. In both cell lines applications of BSO in the maturation medium increased the conversion frequency, i.e. root and shoot emergence, of group A embryos while it enhanced root emergence in group B embryos. Compared to their control counterparts, BSO-treated embryos had normal shoot apical meristems as in their zygotic counterparts. Such meristems were characterized by large apical cells and vacuolated sub-apical cells. They also lacked intercellular spaces, which were present in the apical poles of control embryos where they contributed to cell–cell separation and meristem degradation. Furthermore, storage product accumulation was also improved in the presence of BSO, with protein bodies prevailing over starch. These data show that an oxidized glutathione environment is beneficial for spruce embryo production in vitro.     

In vitro morphogenesis in zygotic embryo cultures of neem (<Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss.)

Immature zygotic embryo cultures of neem yielded highly regenerative cultures, with the response varying with the embryo stage at culture. Early dicotyledonous stage embryos were the most responsive followed by torpedo stage embryos. The embryo cultures differentiated three types of regenerants: somatic embryos (SEs), shoot buds and neomorphs. SEs exhibited morphological abnormalities such as pluricotyledony, fusion of cotyledons and absence of cotyledons. Although these SEs showed secondary embryogenesis, the occurrence of normal dicotyledonous embryos was extremely rare. On MS basal medium 3% of SEs developed a long tap root but a plumular shoot did not appear. However, it was possible to regenerate plantlets from immature zygotic embryo cultures of neem via neomorph formation and adventitious shoot bud formation. The transplantation survival of these plants was more than 80%.Abbreviations BAP 6-Benzylamino purine - CH Casein hydrolysate - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - NAA -Naphthaleneacetic acid - SE Somatic embryoCommunicated by W. Harwood     

Optimization of in vitro bud induction and plantlet formation from mature embryos of Aleppo pine (Pinus halepensis Mill.)

Summary Studies were undertaken to optimize tissue culture conditions for micropropagation of Aleppo pine (Pinus halepensis Mill.) from mature embryos and various explants of the embryo. Over 90% of the embryo explants gave rise to adventitious buds within 4 wk. Intact embryos were the most suitable explants for shoot bud induction. Both isolated cotyledons and hypocotyls produced adventitious buds, but these developed slowly and failed to elongate. N⁶-Benzyladenine (BA) alone at 5.0μM was the most effective cytokinin when added to gelled to gelled von Arnold and Eriksson’s (AE) medium containing 3% sucrose. Adventitious bud development was achieved on hormone-free AE medium, and shoot elongation was optimum on three quarter-strength Bornman’s MCM medium, with 0.1% conifer-derived activated charcoal. Shoots were multiplied on three-quarter strength MCM medium, containing 5μM BA. To induce adventitious roots on the elongated shoots, pulse treatment with 1 mM IBA for 6 h, followed by the transfer of the shoots to sterile peat:vermiculite (1:1) mixture, was beneficial. After acclimatization for 3 to 4 wk under mist, almost all the rooted shoots could be transplanted successfully to the greenhouse, where the plants exhibited normal growth habit. Histologic studies on the ontogeny of adventitious shoot formation from mature embryo explants revealed temporal structural changes in different parts of the explant. Induction of mitotic divisions on the shoot-forming medium resulted in the formation of meristemoids in the epidermal and subepidermal layers of the explant, located initially at both the tips of the cotyledons and the axils of adjacent cotyledons. Shoot buds arising in the axils of adjacent cotyledons were due to new cell division and not to any preexisting meristem.     

Morphological and histological changes during somatic embryo formation from coconut plumule explants

Summary Studies on the development of protocols for the clonal propagation, through somatic embryogenesis, of coconut have been reported for the past three decades, mostly using inflorescence explants, but with low reproducibility and efficiency. Recent improvements in these respects have been achieved using plumular explants. Here, we report a developmental study of embryogenesis in plumule explants using histological techniques in order to extend our understanding of this process. Coconut plumule explants consisted of the shoot meristem including leaf primordia. At day 15 of culture, the explants did not show any apparent growth; however, a transverse section showed noticeable growth of the plumular leaves forming a ring around the inner leaves and the shoot meristem, which did not show any apparent growth. At day 30, the shoot meristem started to grow and the plumular leaves continued growing., At day 45, the explants were still compact and white in color, but showed partial dedifferentiation and meristematic cell proliferation leading to the development of callus structures with a translucent appearance. After 60 d, these meristematic cells evolved into nodular structures. At day 75, the nodular structures became pearly globular structures on the surface of translucent structures, from which somatic embryos eventually formed and presented well-developed root and caulinar meristems. These results allow better insights and an integrated view into the somatic embryogenesis process in coconut plumule explants, which could be helpful for future studies that eventually could lead us to improved control of the process and greater efficiency of somatic embryo and plantlet formation.     

Structure and development of somatic embryos formed inArabidopsis thaliana pt mutant callus cultures derived from seedlings

Summary Seeds of theArabidopsis thaliana mutant primordia timing (pt) were germinated in 2,4-dichlorophenoxyacetic acidcontaining liquid medium. The seedlings formed somatic embryos and nonembryogenic and embryogenic callus in vitro in a time period of approximately two to three weeks. Embryogenesis and callus formation were monitored with respect to origin, structure, and development. Ten days after germination globular structures appeared in close vicinity of and on the shoot apical meristem (SAM). Somatic embryos formed either directly on the SAM region of the seedling or indirectly on embryogenic callus that developed at the SAM zone. Globular structures developed along the vascular tissue of the cotyledons as well, but only incidentally they formed embryos. Upon deterioration, the cotyledons formed callus. Regular subculture of the embryogenic callus gave rise to high numbers of somatic embryos. Such primary somatic embryos, grown on callus, originated from meristematic cell clusters located under the surface of the callus. Embryos at the globular and heart-shape stage were mostly hidden within the callus. Embryos at torpedo stage appeared at the surface of the callus because their axis elongated. Secondary somatic embryos frequently formed directly on primary ones. They preferentially emerged from the SAM region of the primary somatic embryos, from the edge of the cotyledons, and from the hypocotyl. We conclude that the strong regeneration capacity of thept mutant is based on both recurrent and indirect embryogenesis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DIC days in culture - SAM shoot apical meristem     

Comparative anatomy and morphology of Vitis vinifera (Vitaceae) somatic embryos from solid- and liquid-culture-derived proembryogenic masses

Ontogeny of somatic embryos of grapevine (Vitis vinifera) produced from solid- and liquid-culture-derived proembryogenic masses (PEM) was compared using light and scanning electron microscopy. Somatic embryos produced from solid-medium-derived PEM (SPEM) had large cotyledons, little or no visible suspensor structure, and a relatively undeveloped concave shoot apical meristem, whereas those from liquid-medium-derived PEM (LPEM) had smaller cotyledons, a distinct suspensor, and a flat-to-convex shoot apical meristem. The convex shoot apical meristem in LPEM-derived somatic embryos formed as early as the heart stage of development; it was 4-6 cell layers deep and rich in protein. Suspensors persisted in fully developed and mature LPEM-derived somatic embryos. The SPEM-derived somatic embryos exhibited dormancy, as do mature zygotic embryos, which also have a rudimentary suspensor, whereas LPEM-derived embryos were not dormant. We hypothesize that the presence of a persistent suspensor in LPEM-derived somatic embryos modulates development, ultimately resulting in rapid germination and a high plant-regeneration rate.     

On Donor–Acceptor Relations and Growth Correlation in Shoot Development of the Pedunculate Oak Germling

We studied the first cycle of shoot growth of the Quercus robur L. germlings in which the donor–acceptor relations were changed by removing a part of cotyledons, growing in darkness, or removing growing leaf blades. In all cases, the greatest changes in growth and growth correlations were observed in the shoot upper metameres carrying leaf blades and the least, in lower metameres preformed in the acorn embryo. The removal of growing leaves changed the rhythm of shoot growth.