Influence of Cotyledon Excision and Sucrose on Root Formation in Pea Cuttings

Sucrose was supplied to stock plants of Pisum sativum L. cv. Alaska grown at different levels of irradiance. There was no significant effect on the rooting of the cuttings by sucrose supply to intact plants regardless of the irradiance. However, an increase in the number of roots per cutting was obtained at increasing concentrations of sucrose when the stock plants had been grown at 4 W m^?2 and their cotyledons had been removed two days before the cuttings were excised. Cotyledons were removed from stock plants at different times before the excision of cuttings with the intent to regulate the endogenous supply of carbohydrate. The number of roots per cutting was reduced by removal of the cotyledons and this reduction was correlated to the number of days the stock plants had grown without cotyledons as well as to the irradiance pre-treatment. A greater reduction occurred in cuttings from plants grown under 4 W m^?2 than from those grown under 38 W m^?2. The growth of the stock plants and the subsequent stem growth of the cuttings was determined by the irradiance to the stock plants and by the time of removal of the cotyledons. Exogenous supply of sucrose had no effect on the stem growth of the cuttings.     

Influence of the excision of cotyledons on the content of indole-3-acetic acid, growth and flowering of Lupinus albus plants

Excision of both cotyledons of Lupinus albus L. (L. termis Forssk.) seedlings at different morphological stages during the first 8 days after sowing, retarded significantly the growth of 16-day-old seedlings and decreased the content of IAA in the shoots. The effect was more obvious at the early excision (4 days after sowing). A pattern similar to that of control plants with intact cotyledons was attained when cotyledons were excised later than 8 days after sowing. It is believed that the cotyledons control the IAA content of the shoot during the early stages of development. At day 36 flower primordia were observed in controls but not in cotyledon-less plants. At this time the IAA content of the shoot apices and the growth of cotyledon-less plants were the same as in controls except for plants that lost cotyledons 4 days after sowing, which showed similar values at week 11, but did not produce flower primordia. The other cotyledon-less plants produced flower primordia 4–11 days after controls. It is postulated that the cotyledons convey certain factor(s) to the developing axis at an early stage of the development which promote flowering.     

Effect of root formation on 14C-glycine uptake and incorporation into protein by attached and excised cotyledons of Helianthus annuus

Root formation was found to extend the life-span of excised cotyledons of Helianthus annuus L. markedly. Excised cotyledons of 12-day-old plants attained longer life-span, higher root number and total root length than cotyledons excised before or after 12 days. Protein content of attached cotyledons reached a maximum level 12 days after the commencement of germination followed by a decrease. Cotyledons excised 8 days after sowing showed maximum level of protein content 44 days after excision followed by a decrease. The increase was correlated with the full development of roots. The incorporation of ¹⁴C-glycine into protein followed a pattern similar to the protein content, both in attached and detached cotyledons.     

Transport of Benzyladenine and Gibberellin A(1) from Roots in Relation to the Dominance between the Axillary Buds of Pea (Pisum sativum L.) Cotyledons

In etiolated, 5-day-old pea (Pisum sativum L.) seedlings a significantly more intensive growth of buds situated in the axil of the excised cotyledons was observed as early as 4 hours after decapitation and excision of one cotyledon of each pair. If [8-¹⁴C]benzyladenine ([¹⁴C]BA) was applied to roots of intact plants 10 hours prior to such decapitation and excision, significantly higher both total and specific ¹⁴C activities were observed in buds situated on the side of the excised cotyledons as early as 4 hours after decapitation and excision. Although the removal of a substantial part of the root system carried out simultaneously with decapitation and excision of one cotyledon resulted in a decrease in total ¹⁴C activity of buds, nevertheless a higher accumulation of ¹⁴C activity was maintained in buds situated on the side of excised cotyledon. If [¹⁴C]BA was applied to roots of seedlings after they were decapitated and deprived of one cotyledon, both total and specific ¹⁴C activities of buds situated on the side of excised cotyledons were significantly higher as early as the end of uptake of [¹⁴C]BA by roots, i.e. after 10 hours. On the other hand, [1,2-³H]gibberellin A₁ applied to roots of intact and/or decapitated and one-cotyledon-deprived seedlings in the same way as [¹⁴C]BA did not appear in the buds until very much later and only in negligible amounts (i.e.³H activity). This indicates that the release of buds from apical dominance represents an active and selective process which can result from the ability of buds to utilize and/or synthesize only certain growth substances within a certain time interval.     

A double enhancement of greening in the cotyledons of cucumber seedlings by excision and red light

Cotyledons excised without the hypocotyl hook from 6-day-old etiolated cucumber ( Cucumis sativus L. var. Elem) seedlings accumulated a significantly higher amount of chlorophyll than cotyledons excised with hooks or intact cotyledons. It was found that maximum ehancement of greening was achieved after 2 h of dark incubation following excision. Pretreatments with red light effected an additive rise in chlorophyll level in subsequent white light after a dark incubation, suggesting that the effects of excision and phytochrome on greening act independently. Etiolated seedlings were variously dissected before greening and it was found that enhancement occurred only when cotyledons were excised at the level of the hypocotyl hook or above it. Similar results were obtained when the dissected plants were pre-treated with red light.     

GIBBERELLIN RELATIONSHIPS IN THE ‘ALASKA’ PEA (PISUM SATIVUM)

'Alaska’ peas (Pisum sativum L.) grown under a 16-hr photoperiod at 20 ± 1 C and an 8-hr dark period at 16 ± 1 C in their ontogeny exhibit two periods of sensitivity to applied gibberellin (GA), namely, prior to and subsequent to but not during the linear phase of stem elongation. This paper describes experiments conducted primarily with seedlings. Growth-saturating doses of GA, applied to dry seeds before planting (10⁻³ m) and to the shoot tips of 3-day-old seedlings (10 μg), evoked growth rates equal to the growth rate of etiolated seedlings. Sensitivity of seedlings to applied GA decreased with age through the first 2 to 3 weeks of development; by the time seedlings were about 14 days of age and had four elongating internodes they no longer responded to applied GA. As endogenous growth rate diminished late in ontogeny, the plants again became sensitive to applied GA. Growth response was used as a criterion for determining apparent translocation of applied GA. ‘Alaska’ pea seedlings appeared to transport GA, both acropetally from the cotyledons and basipetally from the shoot tip, to all internodes with remaining extension potential. Excision of both cotyledons at any time during the first 9 days of development caused a significant reduction of growth rate, and applied GA did not restore normal growth rate. No evidence was found that the cotyledons supply endogenous GA to the shoot axis in normal seedling development. It is suggested that the normal growth rate of light-grown ‘Alaska’ peas is correlated with the rate of synthesis of GA and that GA is rate-limiting for stem elongation during early seedling development and during the period of decreasing growth rate and onset of apex senescence.     

THE EFFECT OF LIGHT ON THE USE OF THE NITROGEN RESERVES OF GERMINATING SOYBEAN SEEDS

Light affects the mobilization and distribution of several of the storage components of the cotyledons of germinating soybean seeds. The nitrogen content of the cotyledons began to decrease during the first day of germination, continued through day 12 for plants in the light, and day 14 for those in the dark. Cotyledons from both treatments had lost about the same amount of nitrogen by day 14. Plants from both treatments lost about the same amount of dry weight by day 8, but those in the light had taken up nitrogen from the nutrient solution; while those in the dark showed no increase. The plants in the light had higher concentrations of soluble amino nitrogen in the cotyledons than did those in the dark, but the opposite was true for the seedling axis. Aspartate and its amide accounted for half or more of the total free amino acids in all parts of dark-grown plants at 6 and 14 days. In the light-grown plants aspartate and asparagine usually accounted for less than half of the total free amino acids in all plant parts except the cotyledons at 6 and 14 days. Total soluble amino acids were much lower in these plants than those in the dark, excepting the cotyledons.     

THE MOVEMENT OF IAA-C14 IN THE HYPOCOTYL OF PHASEOLUS VULGARIS

The amount of IAA-C¹⁴ transported basipetally through excised hypocotyl sections was strongly affected by the pH of the donor blocks, less so by the pH of the receivers. The effect of donor pH was mostly on uptake. The small amount of acropetal movement was not noticeably affected by pH. Sucrose added to the donor resulted in increased basipetal transport. The time-course of C¹⁴ movement into basal receivers followed a linear course from 1.5 to 3 hr as expected, but there was no net loss from the donors until after 30-45 min. The usual type of velocity calculation, which assumes uptake starting from zero time, would therefore be lower than the true value. Basipetal transport through segments cut from various positions in the hypocotyl and from seedlings of various ages was maximal in 6-8-day-old hypocotyl segments cut 25-30 mm below the cotyledons. Acropetal movement was minimal at all positions of all ages tested.     

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.     

Inter-organ Control of Greening in Etiolated Cucumber Cotyledons

Inter-organ control of greening in etiolated cucumber (Cucumis sativus L. cv. Aonagajibae) cotyledons was investigated. Four- or six-day-old excised or intact etiolated cucumber cotyledons were illuminated under aerobic conditions. Excised cotyledons without hypocotyl hooks produced chlorophyll without a prolonged lag phase and the rate of chlorophyll formation was not depressed if they were illuminated immediately after excision. If the excised cotyledons were incubated in the dark before illumination, chlorophyll accumulation at the end of 6 h of continuous illumination was remarkably lowered as the dark period lengthened, especially in 6-day-old cotyledons. The rapid loss of chlorophyll-forming capacity of excised cotyledons during dark preincubation suggests a stimulatory effect of hypocotyls on the greening in cotyledons. The treatment of excised cotyledons with bleeding sap in the dark for 18 h resulted in the promotion of chlorophyll formation during subsequent continuous illumination. Partial fractionation of bleeding sap with organic solvents and paper chromatography indicates that the active substances showed the same behavior as cytokinins. These facts add weight to the hypothesis that cytokinins from roots flow into cotyledons and stimulate greening.     

Synthesis of DNA in excised watermelon cotyledons grown in water and benzyladenine

Excised watermelon cotyledons were grown in water and benzyladenine, which greatly promotes growth, breakdown of reserves and development of organelles. In order to investigate the involvement of DNA synthesis in these benzyladenine-induced effects, [³H]thymidine was applied continuously (for 3 d) or administered briefly (5 h) to excised cotyledons at various stages of development. Autoradiographic analysis of squashed and sectioned cotyledons showed that both the cytoplasm (mainly in the region of the plastids) and most of the nuclei were labelled. Both types of labelling were promoted by benzyladenine treatment. The highest percentage of labelled nuclei was found in the early stages of growth (first day after excision of cotyledons), long before the burst of enzymatic activities involved in the germination processes. The possible meaning of the increase of nuclear DNA, apart from the normal replicative synthesis preceding cell division, is discussed.Abbreviations BA N⁶-benzyladenine - DNase deoxyribonuclease - EtBr ethidium bromide - FUdR fluorodeoxyuridine - [³H]T [methyl-³H]thymidine     

Turnover of catalase heme and apoprotein moieties in cotyledons of sunflower seedlings

The turnover of catalase apoprotein and catalase heme was studied in cotyledons of sunflower (Helianthus annuus L.) seedlings by density labeling of apoprotein and radioactive labeling of heme moieties. The heavy isotope (50% ²H₂O) and the radioactive isotope ([¹⁴C]5-aminolevulinic acid) were applied either during growth in the dark (day 0-2.5) or in the light (day 2.5 and 5). Following isopycnic centrifugation of catalase purified from cotyledons of 5-day-old seedlings, superimposition curve fitting was used to determine the amounts of radioactive heme moieties in native and density-labeled catalase. Data from these determinations indicated that turnover of catalase heme and apoprotein essentially was coordinate. Only small amounts of heme groups were recycled into newly synthesized apoprotein during growth in the light, and no evidence was found for an exchange of heme groups in apoprotein moieties. It followed from these observations that degradation of catalase apoprotein was slightly faster than that of catalase heme. A degradation constant for catalase apoprotein of 0.263 per day was determined from the data on heme recycling and the degradation constant of catalase heme determined previously to be 0.205 per day (R Eising, B Gerhardt [1987] Plant Physiol 84: 225-232).     

Root Formation by Detached White Mustard (Sinapis alba) Cotyledons

Rooting is shown to occur in excised cotyledons of Sinapis alba when grown in petri dishes on moist filter paper. Cotyledons were excised at intervals from 6 hours after the start of imbibition, when they were yellow, unexpanded and enclosed within the testa, to 27 days after sowing when the cotyledons were green and fully expanded and on plants possessing up to 3 foliage leaves. Rooting generally began 5 or 6 days after excision and was completed dining the following 5 days. The age of cotyledons at t ho time of excision had three effects on rooting: the lag period be-fore rooting began and the period during which rooting took place both increased with age: but the most marked effect was on the total number of cotyledons which were able to form roots, which increased until cotyledon expansion was almost complete, then decreased as the mature cotyledons became older. Optimal rooting was shown by cotyledons detached 8 days alter sowing, when they were half expanded. At this age S5 % of them formed roots between 6 days and 8 days after excision.     

Evidence that Ethylene is not Involved in Red-light-induced Stimulation of Chlorophyll Formation in Etiolated Cucumber Cotyledons

The involvement of ethylene in red-light-induced stimulationof chlorophyll (Chl) formation was studied because one of thered-light effects on Chl formation (the lateappearing effect)interacts with the ethylene effect in 3-day-old excised etiolatedcotyledons of cucumber (Cucumis sativus L. cv. Aonagajibai).Ethylene production by etiolated cotyledons of intact seedlingsin the dark is enhanced by a red-light pulse, but the effectdoes not occur in excised cotyledons. Application of ethylenein the dark to 3-day-old intact seedlings has little effecton Chl formation in the cotyledon during subsequent continuousillumination, although ethylene pretreatment of 5-day-old seedlingssignificantly stimulates Chl formation. Removal of endogenousethylene by mercuric perchlorate [Hg(ClO₄)₂] does not specificallysuppress the red-light action on Chl formation in both attachedand excised cotyledons. Inhibition of ethylene synthesis byaminoethoxyvinylglycine does not affect the red-light effecton Chl formation in excised cotyledons. These facts indicatethat ethylene does not operate as a mediator of red light instimulating Chi formation in either attached or excised cotyledons. (Received December 13, 1981; Accepted March 30, 1981)     

Role of ethylene in chlorophyll degradation in radish cotyledons

The effect of age of radish seedlings on changes in chlorophyll concentration caused by ethylene was examined. Ethylene was produced at 2–4 nl g^?1 h^?1 following excision of cotyledons from 5-to 20-day-old seedlings. The youngest cotyledons maintained this rate, whereas ethylene synthesis declined by as much as 80% during a 24-h period in older cotyledons. The youngest cotyledons continued to accumulate chlorophyll in the dark, but after 7 days cotyledons lost chlorophyll and the proportion of chlorophyll lost increased with age. Ethylene promoted, and norbornadiene inhibited, this loss of chlorophyll; in combined treatments the effects of ethylene and norbornadiene were competitive. The maximal rate of chlorophyll loss occurred in 1μl L^?1 ethylene; extrapolation of the response to concentration indicated that half-maximum loss would occur at 0.005–0.01 μl L^?1 ethylene. In cotyledons from 20-day-old seedlings, chlorophyll degradation occurred mainly after 24 h from excision and transfer to the dark. Chlorophyll degradation during 48 h in the dark was affected by norbornadiene or ethylene applied from 0–24 h or from 24–48 h.     

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.     

Uptake and effects of N-benzyladenine in excised watermelon cotyledons: influence of cotyledon age

Some characteristics of uptake of [8-¹⁴C]N⁶-benzyladenine (BA) by watermelon (Citrullus vulgaris Schrad., cv. Fairfax) cotyledons that were either excised immediately after 24 hours inhibition (day 0) or cultured in the dark for 48 hours on moist filter paper (day 2) have been compared.

The uptake of BA seems to be passive in cotyledons of both kinds. The initial rate of uptake is, however, much slower in day 2 cotyledons. This is probably due to a higher resistance of cell membranes to BA influx. When the day 2 cotyledons are frozen and thawed, so that the membrane barrier is abolished, the amount of BA taken up is the same as in day 0 cotyledons.

In spite of the lower rate of uptake, the physiological effects of BA in day 2 cotyledons are as strong as in day 0 cotyledons and occur with a shorter lag time. Sensitivity to BA seems indeed to start 24 to 48 hours after excision.

     

Gradients of turgor, osmotic pressure, and water potential in the cortex of the hypocotyl of growing ricinus seedlings : effects of the supply of water from the xylem and of solutes from the Phloem

To evaluate the possible role of solute transport during extension growth, water and solute relations of cortex cells of the growing hypocotyl of 5-day-old castor bean seedlings (Ricinus communis L.) were determined using the cell pressure probe. Because the osmotic pressure of individual cells (πⁱ) was also determined, the water potential (ψ) could be evaluated as well at the cell level. In the rapidly growing part of the hypocotyl of well-watered plants, turgor increased from 0.37 megapascal in the outer to 1.04 megapascal in the inner cortex. Thus, there were steep gradients of turgor of up to 0.7 megapascal (7 bar) over a distance of only 470 micrometer. In the more basal and rather mature region, gradients were less pronounced. Because cell turgor ≈ πⁱ and ψ ≈ 0 across the cortex, there were also no gradients of ψ across the tissue. Gradients of cell turgor and πⁱ increased when the endosperm was removed from the cotyledons, allowing for a better water supply. They were reduced by increasing the osmotic pressure of the root medium or by cutting off the cotyledons or the entire hook. If the root was excised to interrupt the main source for water, effects became more pronounced. Gradients completely disappeared and turgor fell to 0.3 megapascal in all layers within 1.5 hours. When excised hypocotyls were infiltrated with 0.5 millimolar CaCl₂ solution under pressure via the cut surface, gradients in turgor could be restored or even increased. When turgor was measured in individual cortical cells while pressurizing the xylem, rapid responses were recorded and changes of turgor exceeded that of applied pressure. Gradients could also be reestablished in excised hypocotyls by abrading the cuticle, allowing for a water supply from the wet environment. The steep gradients of turgor and osmotic pressure suggest a considerable supply of osmotic solutes from the phloem to the growing tissue. On the basis of a new theoretical approach, the data are discussed in terms of a coupling between water and solute flows and of a compartmentation of water and solutes, both of which affect water status and extension growth.     

Effects of auxin,cytokinin, ethrel and cotyledon excision on the peroxidase activity in cotylar buds of decapitated pea (Pisum sativum L.) plants

In decapitated pea plants an increased peroxidase activity ocours after the cotyledon excision in cotylars growing in axillas of cotyledons prior to the growth of excised cotylars. It was found that peroxidase plays an important role in this correlation. Auxin applied on epicotyl stumps inhibited the growth of cotylars and the removal of this inhibition by cytokinin or ethrel was associated with an increased peroxidase activity followed by the growth of cotylars situated in axillas of treated cotyledons.     

Adenosylhomocysteinase and adenosine nucleosidase activities in Lupinus luteus cotyledons during seed formation and germination

The activities of adenosylhomocysteinase (EC 3.3.1.1) and adenosine nucleosidase (EC 3.2.2.7) were assayed in extracts from yellow lupin (Lupinus luteus L.) cotyledons at different stages of seed formation and seedling development. Adenosylhomocysteinase activity was demonstrated in all the cotyledon extracts examined. Its lowest level was found in the dry seeds and the highest, in 4-day-old seedling cotyledons. Extracts from the cotyledons of maturating seeds, dry seeds, and seedlings up to the second day of growth exhibited no adenosine nucleosidase activity. Adenosine nucleosidase activity appeared in the cotyledons of 2-day-old seedlings and its highest level was reached in 4-to 5-day-old seedlings. There is no inhibitor of adenosine nucleosidase in the maturating and dry yellow lupin seeds. No activator of a possible zymogen form of adenosine nucleosidase from maturating or dry seeds occurs in the growing seedlings.