The Effect of Root Formation on the Levels of Protein, Chlorophyll, RNA, DNA and Carbohydrates in Excised Cotyledons of Cucurbita pepo

In attached cotyledons of Cucurbita pepo L. protein, chlorophyll, RNA, DNA and carbohydrates were found to increase to a maximum level 14 days after commencement of germination, followed by a sharp decline thereafter. Cotyledons excised and planted in a nutrient solution 14 days after sowing showed maximum levels of protein, chlorophyll, RNA, DNA and soluble and insoluble sugars 52 days after planting. The increase in these compounds coincides with the full development of the root system. Excision earlier or later than 14 days resulted in lower levels of protein and other compounds. The decrease in the level after 52 days was parallel to the senescing phase of the cotyledons.     

The Effects of Age and Root Formation on the Life-Span of Excised Cotyledons of Cucurbita pepo

Root initiation in Cucurbita cotyledons excised at various ages was studied in nutrient solution and water. The time required for root initiation depends on the age of the cotyledon and on the rooting medium. Nutrient solution favoured root formation. Maximum number of roots, maximal total root length, and maximum life-span were found with cotyledons excised at an age of 14 days. Excision earlier or later resulted in less roots, less total root length, and shorter life-span.     

Histological Study of Adventttious bud Formation on Lactuca Sativa Cotyledons Cultured In Vitro

Abstract

Cyto-histological changes accompanying the formation of adventitious buds in excised cotyledons of Lactuca sativa were studied during the first 12 days after planting in vitro. Prospective proliferating cells can first be recognized, already on the first day after planting, by a marked increase in nuclear and nucleolar volumes, followed on the second day by a burst of cell divisions involving particularly mesophyll cells. Then lignified elements develop together with meristematic center, forming a callus-like tissue in the inner part of the cotyledons. At the third day of culture, the epidermal cells start to divide with a periclinal wall followed by an anticlinal division. In the following days of culture the epidermal cells, which divide mainly with periclinal walls, form layers of cells below the surface, gradually filling up the intercellular spaces. From the 8^th day on, the buds protude above the surface and develops into shoots. These results are discussed in relation to DNA content of nuclei of Lactuca sativa cotyledons and to the time course of cell division and tracheary element formation. The very regular sequence of changes associated with the initiation and development of the bud makes the in vitro culture of Lactuca cotyledons an appropriate System for histochemical and biochemical studies.     

Inhibitory effect of polyamines on the activity of endopeptidase in mung bean cotyledons

The activity of cysteine endopeptidase (EP) in the cotyledons of mung bean seeds increased with time after germination. When cotyledons were excised from the embryonic axis in the course of seedling growth, the activity of EP in the excised cotyledon markedly dropped during the following incubation of 1 d. However, the level of EP protein in excised cotyledons, as examined by immunoblotting, was similar to that in axis-attached cotyledons at the corresponding stage. Thus, it seems that the low activity of EP in excised cotyledons is not due to a decrease in the content of EP protein, but due to a loss of the activity of existing EP. Treatment of attached cotyledons with polyamines (PAs; putrescine and spermidine [Spd]) resulted in a decrease in EP activity, while the same PA-treatment brought about little alteration in the level of EP protein. This indicates that PAs somehow produce an inhibitory effect on the activity of EP. Axis-removal resulted in an accumulation of Spd in the cotyledon. The possibility is suggested that PA, especially Spd, is involved in the inhibition of EP activity in excised mung bean cotyledons.     

Activities of Ribulose Bisphosphate Carboxylase and Phosphoenolpyruvate Carboxylase and C-Bicarbonate Fixation during in Vitro Culture of Pinus radiata Cotyledons

The activities of ribulose bisphosphate carboxylase (RuBPC) and phosphoenolpyruvate carboxylase (PEPC), as indicators of autotrophic and nonautotrophic CO₂ fixation, were measured in excised cotyledons of Pinus radiata D. Don cultured for 21 days under shoot-forming (SF) and nonshoot-forming (NSF) conditions. The activity of RuBPC was found to increase in both SF and NSF cultures during the initial 5 days of culture. However, it leveled off from day 5 to day 10 and subsequently began to decrease until the end of the culture period under the SF conditions. In contrast, in the NSF cultures, RuBPC activity increased until day 15, when it was twofold higher than the maximum activity found in the SF cultures. An increase in PEPC activity of about 2.5 times the level of activity in freshly excised cotyledons was observed during the initial 5 days of culture under the SF conditions. PEPC activity began to decline after day 5 until it reached the level of activity seen in NSF cotyledons by day 15. In contrast, the activity of PEPC did not show any significant increase during the initial 10 days of culture under the NSF conditions. The K_m (phosphoenolpyruvate) of PEPC from SF cotyledons was about 35% higher than that of NSF cotyledons. Cotyledons from two culture periods (days 5 and 15) were incubated for 15 seconds with NaH¹⁴CO₃. The label in the malate and asparatate fractions as a percentage of total ¹⁴C incorporation was 3 times higher in the SF cotyledons than in the NSF cotyledons. A higher incorporation of ¹⁴C into products of photosynthesis under the NSF conditions was also observed.     

Influence of the Embryonic Axis on Protein Hydrolysis in Cotyledons of Cucurbita maxima

Four-day time course studies of the hydrolysis of cotyledonal storage protein were conducted on intact seeds, seed cotyledons detached from their embryonic axes and on detached cotyledon pairs germinated in the presence of three excised embryonic axes of Cucurbita maxima Duch., cv. Chicago Worted Hubbard. Detached cotyledons germinated alone showed little hydrolysis of the storage protein. However, the amount of protein hydrolysis of the detached cotyledon pairs germinated in the presence of three excised embryonic axes was comparable to the amount hydrolyzed in the cotyledons of intact germinating seeds. Visual growth differences among these treatments were also evident. The size and yellow color intensity of the fourth day treatments were shown to increase in the following order: detached cotyledon pairs alone, intact seedlings, detached cotyledon pairs in the presence of three excised axes. The growth of the hypocotyl and radical was also modified by removal of the cotyledons. These findings suggest that storage protein degradation and cotyledonal growth are controled by the axis. They also indicate that the cotyledons have some influence on the growth of the axes. Time-course studies were made on the hydrolysis of storage protein in the cotyledons of squash and on the distribution of the hydrolytic products during the germination of light- and dark-grown plants. The storage protein was not hydrolyzed during the first 24 hours. It was hydrolyzed at a uniform rate from 1 to 5 days and at a slightly decreased rate from 5 to 7 days. Most of the hydrolytic products were transported to the axial tissue. Proteinase activity in the cotyledons rapidly increased during germination to a maximum level at 2 to 3 days. This was followed by a decline to about the initial value after 7 days.     

The Change in Arginine Levels and the Metabolism of Urea and Ornithine in Cucurbita moschata Seedlings

During germination a decrease in both soluble and protein argmine occurs in pumpkin cotyledons (Cucurbita moschata Poir.) and this decrease parallels the decrease in total nitrogen. Arginine is readily metabolized to urea and ornithine and the metabolism of the latter compounds were studied by allowing cotyledons of intact plants to absorb urea^?14C and by incubating cotyledon discs with ornithine-2^?14C. Of the urea absorbed, 90 % of the label was metabolized to ¹⁴CO₂ with little free urea remaining in the plant. Urease activity in the cotyledons reached a maximum at 4 to 6 days of germination. Ornithine was extensively metabolized with 37 % of the label being incorporated into protein by 3 h. Soluble and protein arginine accounted for 23 % of the label suggesting that Krebs-Henseliet cycle enzymes were present.     

Changes in endogenous levels of three cytokinins during development of excised watermelon cotyledons

We have determined by an immunological method the endogenous levels of three cytokinins: dihydrozeatin riboside (DHZR), transzeatin riboside (tZR) and isope-ntenyladenosine (IPA) in watermelon (Citrullus vulgaris Schrad., cv. Fairfax) cotyledons that were either attached to the seedling or excised from the seed after imbibition and then grown on water. Both seedlings and cotyledons were grown either for 5 days in continuous light or for 3 days in the dark and 2 days in light. Our aim was to verify whether endogenous cytokinin levels are lower in excised than in attached cotyledons as could be expected since excised cotyledons are much more sensitive to exogenous cytokinin application. The levels of the three cytokinins were very low immediately after imbibition, but gradually increased during the following days. They were higher in excised cotyledons after 5 days of culture in the dark than in cotyledons of the same age that had developed on the seedling. Dihydrozeatin riboside was by far the most abundant of the three cytokinins in cotyledons as well as in the hypocotyl and the root.
Irradiation reduced the level of DHZR, negating the concept that light promotes cotyledon development by increasing endogenous cytokinins. Transzeatin riboside when supplied exogenously, stimulated cotyledon development at a lower concentration than the other two cytokinins. Exogenous supply of ben-zyladenine (BA) induced a strong increase in endogenous tZR already after 24 h.     

Protein Breakdown and Formation of Protease in Attached and Detached Cotyledons of Phaseolus vulgaris L

In contrast to earlier reported results of similar experiments in peas, in which almost no increase in protease activity occurred in incubated detached cotyledons, we report here an increase in protease activity in both attached and detached bean cotyledons. Detached bean cotyledons showed continually increasing protease activity up to the 12th day, while that in attached cotyledons declined after 6 days. The free amino acid level in detached cotyledons reached a maximum at the 11th day; protease formation leveled off after 50% of the original seed protein was digested. These data suggest that high free amino acid levels may inhibit protease formation.     

The Effects of Benzylaminopurine on Growth and 14Carbon Translocation in Excised Mustard Cotyledons

When localized areas of blades of recently excised mustard cotyledons were fed with ¹⁴CO₂ it was found that the fixation products passed rapidly into the veins and then were translocated to the petiole. Since other cotyledons similarly treated subsequently rooted at the petiole base, this suggested that it acted as a sink for assimilates. Treatment of excised cotyledons with benzylaminopurine (BAP) resulted in the enhancement of blade growth and the suppression of root initiation and development. Quantitative determinations of labelled assimilate in both the cotyledon blade and the terminal 2 mm portion of the petiole were made. It was found that cotyledons treated with BAP at a concentration high enough to suppress completely root initiation had a higher level of radioactivity in the petiole base than the terminal segments in untreated petioles, whether expressed as specific activity or as a percentage of the total radiocarbon fixed. BAP-treated cotyledons also fixed consistently higher levels of ¹⁴CO₂, probably due to an increase in photosynthetic area. The results suggest that BAP alters the pattern of differentiation at the base of the petiole rather than the polarity of movement of assimilates to the petiole base.     

Effects of Ammonium and Potassium Ions on Some Physiological and Biochemical Processes of Excised Cucumber Cotyledons

Incubation of excised cucumber cotyledons (Cucumis sativus L.) with NH₄Cl solutions exceeding 0.001 M inhibited their greening, fresh weight increases, and incorporation of ¹⁴C-leucine into insoluble N compounds. The absorption of ¹⁴C-leucine during incubation and retention of moisture by the excised cotyledons after incubation were greatly diminished by the NH₄Cl treatments. Treatment with KCl solutions of the same concentrations as the NH₄Cl stimulated the greening, fresh weight increases, and the absorption and incorporation of ¹⁴C-leucine. Desiccation of cotyledons stored at 5°C for 10 days was inhibited by dilute KCl solutions. The toxicity of NH₄Cl was alleviated by KCl solutions at low concentrations.     

Auxin-dependent breakdown of xyloglucan in cotyledons of germinating nasturtium seeds

Rapid mobilisation of storage products, including xyloglucan, in cotyledons of germinating nasturtium (Tropaeolum majus L.) normally starts about 7–8 d after imbibition and growth of the seedling at 20–25° C. Levels of activity of endo-1,4--glucanase (EC 3.2.1.4) in cotyledons, as assayed viscometrically with xyloglucan as substrate, varied in parallel with the rate of breakdown of xyloglucan. When cotyledons were excised from the seedling axis and incubated on moist filter paper at any point before 7 d, the catabolic reactions which normally occurred in the intact seedling were suspended. If, however, cotyledons excised at 8 d were incubated in 10^–6 M 2,4-dichlorophenoxyacetic acid, a rise in endo-1,4--glucanase (xyloglucanase) activity was observed and a sharp decrease in fresh and dry weight as well as xyloglucan levels ensued at rates comparable to those observed in cotyledons attached to the seedling. Neither gibberellin nor kinetin treatments promoted xyloglucan breakdown or enhanced xyloglucanase activity. Addition of auxin to excised cotyledons before 7 d did not evoke premature breakdown, indicating that the tissue became receptive to auxin only at this time. The triggering process took place in darkness and was unaffected by various light-dark cycles. It is concluded that the sudden degradation of xyloglucan which occurs in nasturtium seeds about a week after germination begins is the result of enhanced activity of a depolymerizing xyloglucanase, this activity being evoked by auxin originating in the emerging seedling axis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,3-D 2,3-dichlorophenoxyacetic acid - GA₃ gibberellic acid - kDa kilodalton The authors are pleased to acknowledge the technical assistance of Alexander Marcus and valuable discussions with Dr. Vladimir Farkas. This study was supported by a scholarship to A.H. from the Deutsche Forschungsgemeinschaft (FRG) and a grant to G.M. from the Natural Sciences and Engineering Research Council of Canada.     

Jasmonic acid inhibition of in vitro shoot organogenesis in Pinus radiata cotyledons

The effect of jasmonic acid (JA) onde novo shoot formation in excised cotyledons of radiata pine (Pinus radiata D. Don), was examined. JA had no effect on shoot-forming (SF) tissues at concentrations up to 10⁻⁶ mol · L⁻¹. At concentrations greater than that, JA caused a reduction in the number of shoots formed, as well as the lengths of the shoots and fresh and dry masses of the tissues. Reciprocal transfer of excised radiata pine cotyledons from a SF medium with 10⁻⁵ mol · L⁻¹ JA to a SF medium without JA and vice versa showed that any exposure of the cotyledons to JA either during the shoot induction phase (days 0-21) or the shoot development phase (beyond day 21) led to a reduction in shoot formation. However, the JA effect was significantly less if the cotyledons were not exposed to JA during the initial 10 days in culture; indicating that the JA effect was mainly during shoot primordia formation and the subsequent development into shoots.     

Control of Protein Hydrolysis in the Cotyledons of Germinating Pea (Pisum sativum L.) Seeds

The effects of removal of the shoot or whole axis on the levelsof total, protein, and TCA-soluble nitrogen and on proteaseactivity in cotyledons during germination of garden pea (Pisumsativum L ) seedlings grown in the light have been examined. Removal of the shoot 1 week after soaking the seed caused areduction in the rates of protein hydrolysis and of nitrogentransport from the cotyledons and an increase in the level ofsoluble nitrogen When the entire axis was excised after 4 or9 days there was a great reduction in protein hydrolysis whilethe level of soluble nitrogen remained the same as in de-shootedplants. In the intact plant, proteolytic activity of cotyledon extractsrose to a peak about 15 days after soaking of the seed and thenfell rapidly This fall coincided with a decrease in water contentand in oxygen consumption by the cotyledons. Removal of theshoot or entire axis led to a much smaller and more gradualincrease in protease activity and the subsequent decline inactivity of the enzyme and senescence of the cotyledons werealso delayed. It is concluded that control of protein hydrolysis in pea cotyledonsis not mediated through the level of protease enzymes, as indicatedby the proteolytic activity of tissue extracts, or by the amountof soluble nitrogen compounds accumulated. Protease activityseems to be controlled by the shoot and to be closely linkedto senescence of the cotyledons Protein hydrolysis and transportof nitrogen to the axis, on the other hand, are affected bythe presence of both shoot and root and the axis appears toexert independent control on each of these processes.     

Cytokinins in Germinating Seeds of Phaseolus vulgaris L. I. Changes in Endogenous Levels Within the Cotyledons

Ethanolic extracts from the cotyledons of mature dry Phaseolusvulgaris L. seed yielded cytokinin-like activity which co-chromatographedwith zeatin and ribosylzeatin. Under conditions which stimulatedgermination and cotyledon expansion, the level of these cytokininsdecreased rapidly in both intact embryos and excised cotyledons.In the excised cotyledons the decrease was continuous, resultingin very low levels of cytokinin being detected after 4 daysof incubation. With the embryonic axis present, however, theinitial decrease was arrested and reversed after 3 days. Thissuggests that the cotyledons do not synthesize cytokinins butthat these hormones are imported from the embryonic axis, particularlyonce radicle growth is well under way. Phaseolus vulgaris, bean, cotyledons, cytokinins, germination     

The effects of light and cotyledon age on growth and root formation in excised cotyledons of Sinapis alba L.

Summary Root formation on excised mustard cotyledons was found to be determined by cotyledon age at excision and by light. Light treatment after excision enhanced root production in cotyledons excised 6 hours to 6 days after the start of imbibition and was essential for root production in cotyledons older than 6 days at excision. Three or more 12 hour light periods after excision were sufficient to induce root production in almost 100% of 7 day old cotyledons. The interposition of up to 5 days of darkness between excision and subsequent culture in the light did not reduce the final rooting percentage but delayed the onset of rooting by a time equivalent to the length of the dark period. The dry weight values of the cotyledons were directly related to the total light energy received after excision.The age and light effects on rooting may be mediated by their effects on the rate of breakdown and on the total levels of available reserves present in the cotyledons.     

Adventitious root induction inCorylus avellana L. cotyledon slices

Summary An experimental rooting system was developed to study in vitro adventitious root formation in hazelnut cotyledons. Experiments involveda) assay of several culture media,b) use of different developmental status of the seeds (germinated and ungerminated),c) cotyledons subjected to various light regimes, andd) different size of cotyledons slices. It was observed that higher rooting was induced in cotyledonary portions of 5- or 7-mm thickness (250 and 350 mm³, respectively) cultured on half-strength basal medium supplemented with 50 μM indole-3-butyric acid and 5 μM kinetin. Rooting was affected by light and the developmental state of seeds. Preinitiation, initiation, and root manifestation stages were defined according to specific culture periods and in relation with morphologic and histologic changes. The first histologic changes (cell division and root primordia induction) were observed after 12 days in culture. At 30 days of culture in rhizogenic medium root primodia were fully differentiated with well-developed vascular tissues.     

Endogenous indole-3-acetic acid during adventitious root formation inPopulus £canadensis Moench.

Indole-3-butyric acid (IBA), phenylacetic acid (PAA) and naphthaleneacetic acid (NAA) were applied at a concentration of 10^-4 mol dm^-3 to stem cutting bases ofPopulus x canadensis Moench. During adventitious root formation, the content of indole-3-acetic acid (IAA) in cutting bases was estimated using the fluorimetric method. In the control variant, a rapid increase in endogenous IAA appeared after 24-h cultivation followed by gradual decrease during the following days. In contrast, the variants treated with IBA, PAA, and especially NAA exhibited firstly a decrease in endogenous IAA content and only afterwards an increase, reaching a maximum 48 h after excision. As root regeneration proceeded gradually, a decrease in the level of endogenous IAA occurred in all treatments. The first adventitious roots appeared in all treatments after 216-h cultivation.     

Seed and soil: sources of inoculum for the colonisation of the foliage of solanaceous hosts by Pseudomonas solanacearum

Pseudomonas solanacearum was transmitted from contaminated seed to the cotyledons of capsicum (Capsicum frutescens) at 92, 73 and 60% r. h. and to the cotyledons of tomato (Lycopersicon esculentum) seedlings at 92% r. h. Subsequent epiphytic colonisation of the true leaves of capsicum occurred at 92 and 73% r. h. An increase in the population on capsicum cotyledons was detected at 92 and 73% r. h. but only at the higher r. h. was an increased population of P. solanacearum associated with the colonisation of the true leaves. Lesions developed on the true leaves of capsicum at 92% r. h. Transmission of P. solanacearum from capsicum seeds was affected by inoculum concentration, occurring at an infestation level of ∽ 10³ propagules seed^-1 but not at 50 propagules seed^-1. Pseudomonas solanacearum was detected on the cotyledons of capsicum held at 98% r. h. after germination of seed in soil infested with 2 × 10⁸ propagules g^-1 soil; lesions were detected on cotyledons 11 days after planting and invasion occurred in 10 stems and one root of the 20 seedlings sampled. The movement of the pathogen from the soil to the seedlings was affected by the level of soil infestation. Pseudomonas solanacearum was detected in four pairs of cotyledons, two stems and one root of the 36 seedlings sampled from soil infested with 10⁷ propagules g^-1 soil but it was not isolated from seedlings sampled from soil infested with 10⁵ or 4 × 10³ propagules g^-1 soil. Leaf and stem prints demonstrated the epiphytic nature of this organism on the cotyledons and stems of seedlings.     

Growth and synthesis of nucleic Acid and protein by excised radish cotyledons

Nutritional and light requirements for growth and synthesis of RNA, DNA, and protein by cotyledons excised from 5-day-old seedlings of Raphanus sativus L. were investigated, and the course of synthesis was followed through the cell cycle. The minimum requirements for a net increase in nucleic acid and protein were sugar, nitrate, and light. The cotyledons used nitrite at low concentration, but not ammonium ion. Light was required for preliminary steps in synthesis of RNA, DNA, and protein, but the actual polymerization reactions occurred in the dark. The cotyledons contained sufficient endogenous growth factors for about half of the cells to complete 1 cycle on a medium of 1% sucrose, 80 mm KNO₃. The increase in DNA was limited to about 50% and was accompanied by a comparable increase in cell number. Fresh weight, RNA, and protein tended to increase in proportion to DNA. Growth of the isolated cotyledons commenced with cell enlargement. RNA began to increase after about 4 hours, DNA after about 12. The major increase in protein also began at about 12 hours. The maximum rate of increase for all 3 occurred between 12 and 16 hours. Cell counts indicated that by 28 hours most of the cells which had replicated DNA had also completed cell division.