The Effect of Growth Conditions and Origin of Tissue 5Phaseolus vulgaris L.)

Callus cultures isolated from various somatic tissues and anthertissue of Phaseolus vulgaris seedlings on a defined growth mediumcontained few diploid cells. The proportion of diploid cellsdid not alter as cultures lost their ability to form vasculartissue. Meristematic cells of roots initiated after transferto induction medium were diploid. All cultures lost their morphogeneticpotential after five to seven subcultures except anther calluswhich formed vascular tissue over a prolonged period of cultureon maintenance medium. After six subcultures anther callus containedmore polyploid cells than somatic cultures. Callus isolated from bean hypocotyl tissue in the presence ofcoconut milk consisted mainly of diploid cells and retainedits morphogenetic potential for a greater number of subculturesthan callus grown on defined medium. Transfer of callus isolatedon the defined medium to medium containing coconut milk increasedthe proportion of diploid cells and prevented further loss ofinorphogenetic potential. An equivalent concentration of cytokininto that in coconut milk prevented the loss of potential butdid not affect the ploidy of the cultures.     

Condensed Tannin Formation by Agrobacterium rhizogenes Transformed Root and Shoot Organ Cultures of Lotus corniculatus

Root cultures of Lotus corniculatus L. cv. Leo transformed withAgrobacterium rhizogenes (C58Cl-pRi15834) grew rapidly in liquidmedium when cultured in the dark and produced large numbersof shoots when illuminated. The shoots, which could be regeneratedto produce fertile plants, were maintained in liquid mediumas shoot-organ cultures The accumulation and cellular distribution of condensed tanninswas determined during the growth of these root and shoot organcultures and in primary callus from non-transformed explants.Root and shoot cultures predominantly accumulated insolublepolymeric tannins which yielded both cyanidin and delphinidinon hydrolysis at ratios equivalent to control plants. Methanol-solublevanillin-positive compounds were isolated but no free oligomericproanthocyanidins, monomeric flavans or dihydroflavonols weredetected in these extracts. Condensed tannin accumulation waslinearly related to root growth and had a similar spatial distributionin ‘tannin’ cells in roots and leaves as comparedto control plants. Tannin-containing cells were absent frommeristematic cells of the root tip and root/shoot interface.Primary callus cultures failed to accumulate condensed tanninson media containing auxins, and exogenously supplied auxinswere found to inhibit tannin accumulation by transformed rootand shoot cultures Key words: Lotus corniculatus, Agrobacterium rhizogenes, hairy roots, condensed tannins, shoot and root cultures.     

Alternating Cytokinins in Multiplication Media Stimulates in Vitro Shoot Growth and Rooting of Eucalyptus globulus Labill.

Shoots of Eucalyptus globulus Labill. cultured on shoot multiplicationmedia containing, on alternate subcultures, 6-benzylaminopurine(BAP) or 6-furfurylaminopurine (kinetin), showed better growththan cultures in which either of the cytokinins was used continuously,or both were used in an equimolar mixture. When BAP was usedcontinuously in the medium (i.e. in every subculture), shootsmultiplied but remained stunted and leaves became red and abscised.Kinetin or 6-dimethylallyaminopurine (2iP) used continuouslyin the medium induced very low multiplication but the shootsdid not become red nor did the leaves abscise. Shoots takenfrom multiplication medium containing BAP and placed on rootingmedium with 10 µM indole butyric acid (IBA) produced fewroots and often died while on the rooting medium. In contrast,shoots from the multiplication medium containing kinetin producedmore roots and remained healthy during the passage on the rootingmedium.Copyright 1994, 1999 Academic Press Eucalyptus globulus, Tasmanian bluegum, cytokinins, micropropagation, in vitro rooting     

In vitro Propagation of Red Cabbage (Brassica oleracea L. var. capitata)

By manipulation of various growth regulators and physical conditions,plants have been regenerated from excised roots, stem segments,cotyledons, leaves, and callus cultures of red cabbage (Brassicaoleracea var. capitata) grown under in vitro conditions. Shootbuds were induced on isolated root segments (1 cm long) culturedon Murashige and Skoog's medium and the frequency of bud formationwas greatly enhanced by the addition of kinetin (0.5 part 10^–6).Callus obtained from the seeds, cotyledons, and hypocotyl segmentscultured on a medium fortified with 2,4-D (1 part 10^–6),kinetin (0.1 part 10^–6), and coconut milk (10%, v/v) hasbeen repeatedly subcultured. The callus is slow growing, andon transference to a kinetin (2 parts 10^–6) and IAA (2parts 10^–6) medium underwent morphogenesis to give riseto plants. The significance of the propagation of red cabbageby in vitro culture is pointed out.     

Effect of Osmotic Stress Upon Endogenous Hormone Levels in Euphorbia lathyrus L. and Vicis faba L.

Ethylene concentrations rise in stems, lacunae and roots ofEuphorbia Iathyrus subjected to osmotic stress. In contrastto other species, however, no such changes are observed in leaves.Similarly, ABA concentrations rise in the leaves of osmotically-stressedplants but little change was observed in roots. In no case wasthere any strong evidence for the existence of a threshold ofosmotic potential above which a response will not occur. Instead,marked increases in concentrations of both ABA and ethylenewere obtained in response to osmotic stresses which did notresult in significant changes in water saturation defecit (WSD)during the experiment. In Vicia faba, time course studies at constant osmotic potentialshowed that ABA concentrations increased with duration of stressand increased WSD but the effect was most marked in youngertissues for a given level of stress. Concentrations of boundABA showed a similar pattern with time but here the change wasleast marked in the younger tissues. Euphorbia Iathyrus, Vicia faba, endogenous growth regulators, osmotic stress     

Changes in the Levels of Endogenous Growth Regulators during Development of the Flowers of Chrysanthemum morifolium

Gibberellin-like substances and an auxin similar to IAA weredetected by bioassays in extracts of flowers of Chrysanthemummorifolium. The activity of these substances was shown to reacha maximum early in the development of the flower when its relativegrowth-rate was at a maximum, and then to decline with the relativegrowth-rate. The leaves of lateral flowering shoots were found to containgibberellins similar to those detected in the flowers whilea different gibberellin, which appeared to decrease in activitywith the age of the shoot, was detected in the stem. An auxinsimilar to indol-3yl-acetic acid (IAA) was also detected inthese stems. Growth-promoting substances were not detected inthe old stems and leaves from the main shoot. Gas-liquid chromatographyrevealed the presence of a number of additional gibberellinsin the flowers. The chemical nature of the growth substances is discussed inrelation to their biological and chromatographic behaviour.     

NUTRITIONAL AND MORPHOGENETIC INVESTIGATIONS ON CALLUS CULTURES OF NEOMAMMILLARIA PROFILERA MILLER (CACTACEAE)

Neomammillaria prolifera (Cactaceae), when grown on Murashige and Skoog's medium supplemented with fresh coconut milk, showed very little growth. Various concentrations and combinations of growth regulators which did not cause callusing had no apparent effect on the normal growth rates of intact plants. Healthy green calli obtained on a 2,4-D and kinetin-containing medium exhibited extremely fast growth and very specific growth requirements. Relatively high amounts of 2,4-D (10–20 mg/liter), kinetin (1–2 mg/liter), and coconut milk (20–60%) were required at all times for continued proliferation of callus on subculturing. Moreover, the callus was very tolerant to extremely high concentrations of other growth regulators (IAA, NAA, IBA, and GA up to 100 mg/liter) in the presence of 2,4-D and coconut milk. These substances could not replace 2,4-D for callusing or continued growth of callus. It was not possible to establish root cultures or to induce callusing of roots. Attempts to induce differentiation in callus were unsuccessful, except for sporadic root initiation in some cultures. A comparison of these results with similar studies on other succulents demonstrates some basic physiological similarities among this group of plants.     

The Relationship of Abscisic Acid to Nitrate Reductase Activity in the Potato Plant

Abscisic acid (ABA) at 3.8 µM suppressed both in vivoand in vitro nitrate reductase activity in roots, stems andleaves of potato plants grown in solution culture. Suppressionwas maximal between 24 and 48 h, followed by recovery of activityat 72 h in roots and leaves and at 96 h in stems. Removal from ABA after 24 h resulted in complete recovery ofnitrate reductase activity in roots by 24 h and partial recoveryin leaves. ABA treatment enhanced nitrate accumulation in roots,decreased that of leaves, but had no effect on stem nitratecontent. ABA enhanced decay of the enzyme following nitrate removal;by 7 h activity in roots was 22.5% of the initial value comparedto 55% in the control. ABA showed a less drastic effect on lossof activity in leaves and stems. These results indicate thatABA suppression of nitrate reductase activity is not dependenton nitrate uptake, and although it reduced leaf nitrate contentthere was no clear relationship between tissue nitrate levelsand the ABA response. (Received September 13, 1984; Accepted July 1, 1985)     

Investigations on the Growth and Metabolism of Plant Cells: V. Tumorous Growth in Relation to Growth Factors of the Type found in Coconut

1. It has been shown that aqueous extracts of plant tumours,induced by Agro-bacterium tumefaciens (Smith and Townsend) onBryophyllum and Kalanchoe, will act in place of coconut milkusing the tissue-culture procedures previously described inthis series of papers. 2. In a large number of experiments it has been shown that tumoursof this kind yield extracts which have a growth-promoting effectsimilar to that of coconut milk. This effect may be enhancedby, though it is distinguishable from the effects of, addedcasein- hydrolysate in the basal medium. The activity of thetumour extracts was consistently greater than the activity ofextracts of stems and leaves of the same plants and of normal,non-tumorous plants. 3. Partial fractionation of the tumour extracts has shown thatactivity was concentrated in the alcohol extracts, and to alesser extent in the water extracts. Activity was completelylacking in the ether extracts. 4. The effect of coconut milk, which is replaceable wholly orin part by tumour extracts, is primarily an effect on cell divisionin the carrot tissue. 5. The bearing of these results on tumorization in plants isdiscussed.     

Effect of inoculation method and plant growth medium on endophytic colonization of sorghum by the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

A study was conducted to determine the effect of inoculation method and plant growth medium on colonization of sorghum by an endophytic Beauveria bassiana. Colonization of leaves, stems, and roots by B. bassiana was assessed 20-days after application of the fungus. Although B. bassiana established as an endophyte in sorghum leaves, stems, and roots regardless of inoculation method (leaf, seed, or soil inoculation), plant growth medium (sterile soil, non-sterile soil, or vermiculite) apparently influenced colonization rates. Seed inoculation with conidia caused no stem or leaf colonization by the fungus in non-sterile soil but did result in substantial endophytic colonization in vermiculite and sterile soil. Leaf inoculation did not result in root colonization, regardless of plant growth medium. Endophytic colonization was greater in leaves and stems than roots. Endophytic colonization by B. bassiana had no adverse effects on the growth of sorghum plants. Leaf inoculation with a conidial suspension proved to be the best method to introduce B. bassiana into sorghum leaves for plants growing in either sterile or non-sterile soil. Further research should focus on the virulence of endophytic B. bassiana against sorghum stem borers.     

The Effect of Abscisic Acid on Amino Nitrogen and Protein Content of Potato Plants in Relation to the Inhibition of Nitrate Reductase Activity

Treatment of potato plants grown in nutrient solution with 3.8µM ABA resulted in reduced soluble protein in roots andin leaves at 24 h, but not in stems. This treatment reducedin vivo nitrate reductase activity in all organs for about 48h with the most pronounced reduction occurring in the roots.Excised root and leaf segments from plants treated with ABAfor 24, 48 and 72 h absorbed significantly more ¹⁴C leucine,compared to the control but the percent incorporation into proteinwas not altered in roots. In response to ABA total free amino nitrogen in leaves was lowerat 5 and 72 h and in stems at 72 h. Amino nitrogen content ofroots was enhanced by ABA at 5, 24 and 72 h due to generallyhigher levels of aspartate, serine, glutamate, proline and ammonia.There was no consistent relationship between ABA suppressionof nitrate reductase activity and ammonia or specific aminoacid (except proline) levels in leaves and stems. The increasedfree amino nitrogen levels in response to the hormone may bethe result of impaired NO₃– reduction rather than thecause. The results of protein synthesis studies and solubleprotein content suggest that ABA inhibition of nitrate reductaseis not due to general inhibition of protein synthesis and mayinvolve specific inhibition of nitrate reductase protein synthesis. ¹ Contribution No. 684, Department of Plant Science, Universityof Manitoba.     

Occurrence of L-DOPA and dopamine in plants and cell cultures ofMucuna pruriens and effects of 2,4-d and NaCl on these compounds

The development of the L-DOPA-content of roots, stems and leaves ofMucuna pruriens during growth of the plants is described. Besides L-DOPA, the leaves, but not the stems and the roots, also contain the related catechol dopamine. The time course of dopamine accumulation is compared to that of L-DOPA. In cell suspension cultures ofM. pruriens dopamine can be detected as well. Its level is strongly increased by addition of the growth regulator 2,4-d to the medium, a condition that suppresses cell growth and L-DOPA-accumulation. Dopamine induction appears to be a specific metabolic effect of 2,4-d. Salt stress, as caused by the addition of NaCl, gives no induction of dopamine formation, whereas L-DOPA is released into the medium.     

Stomata of Micropropagated DelphiniumPlants Respond to ABA, CO2, Light and Water Potential, but Fail to Close Fully

Morphological and physiological characteristics of micropropagatedplants of Delphinium cv. Princess Caroline were studied. Leavesproduced in vitro showed poor control of water loss which appearsto result from restricted responses by stomata and not frompoor cuticular development. Stomata of leaves produced in vitrowere larger and more frequent than those produced during acclimatization.Despite the fact that stomata from isolated epidermis of leavesproduced in vitro reduced their apertures when exposed to turgor-reducingtreatments, they did not close fully. This, together with highstomatal frequencies might explain the poor control of waterloss shown by intact leaves produced in culture when exposedto dry air. While leaves from acclimatized plants showed almostcomplete closure with ABA, low water potentials, darkness andCO₂, stomata from leaves produced in vitro reduced their apertureswhen exposed to those factors, but only to a limit. Therefore,stomata from leaves cultured in vitro seem to be partially functional,but some physiological or anatomical alteration prevents themfrom closing fully. Stomata from leaves produced in vitro wereparticularly insensitive to ABA which appears to be partly associatedwith the high cytokinin concentration in the culture medium.In the long-term, this stomatal insensitivity to ABA might contributeto plant losses when micropropagated plantlets are transferredto soil. Key words: Micropropagation, stomatal physiology, dehydration, PEG, ABA, BAP, darkness, CO₂, Delphinium     

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.     

Regeneration of plants from Antirrhinum majus L. callus

Somaclone production in Antirrhinum majus plants by regeneration of plants from callus cultures has been achieved using three types of explant tissue. Regeneration from mature stem internode-derived callus was extremely poor. Callus derived from seedling shoot tips could be induced to form new shoots in six of seven cultivars tested. Regeneration was achieved in all seven cultivars when callus was produced from segments of hypocotyl and was most effective using agar-solidified medium containing 0.25 mgl^-1 naphthoxyacetic acid + 10% coconut milk. In this case, five of the cultivars produced shoots directly, one produced leaves from the petioles of which new shoots emerged, and one regenerated plants chiefly through the production of embryoids.     

Morphogenesis in Stem-callus Cultures of Convolvulus arvensis L

Convolvulus arvensis stem explants form callus when inoculatedon to 0.5 mgm/1 2, 4-D, 15 per cent coconut milk, and on to1 mgm/1 kinetin, 15 per cent coconut milk. In the latter casenumerous shoots are formed on the callus. Callus formed on explantson 0.05 mgm/1 2, 4-D, 15 per cent coconut milk formed shootswith in-creasing vigour when transferred to a medium (1) inwhich the 2, 4–D concentration is lowered by one-tenth,(2) which contains no auxin but kinetin at 1 mgm/1, (3) whichcontains kinetin at 1 mgm/1 and 15 per cent coconut milk. OnNAA/ kinetin media shoots may form in which the intenode developmentis suppressed, giving compressed shoots. The capacity of callusto form shoots may be retained or lost through repeated sub-culture.Roots are formed erratically. The results are discussed, particularlywith regard to the loss of morphogenetic potential and whetherthis is reversible.     

In Vitro Culture of Orobanche aegyptiaca

Calli derived from seedlings of the parasitic plant Orobancheaegyptiaca were grown on enriched nutrient agar. Growth wasenhanced by the addition of 0.1 mg 1^–1 NAA and 5 % coconutmilk. The calli developed two kinds of elongated protrusions:thin ones which resemble the young root radicle and thick oneswhich resemble the adventitious roots of Orobanche plants. Bothformed pad-like structures when touching the substrate. In liquidculture, some of the calli were observed to surround isolatedcultured tomato roots. Orobanche aegyptiaca Pers., in vitro culture, germination, coconut milk, root parasite     

The Application of the Modified Sine Rule to Tension Wood Production and Eccentric Growth in the Stem of Crack Willow (Salix fragilis L.)

One-year-old shoots of S. fragilis were placed on speciallyprepared frames and grown at various angles from the uprightposition for six-and-a-half months. At the end of this periodthe stems were found to have made greater growth on the upperside than the lower, and the majority of the fibres of the upperxylem were of the gelatinous type. The degree of eccentricityand the amount of gelatinous fibres produced were clearly shownto reach a maximum when the stems were displaced by 120°from the upright position. This provides evidence that in stems,as in roots, geotropic responses are not proportional to thesine of the angular displacement from the vertical, but followa modified sine relationship. These results are considered toprovide support for the hypothesis that statolith graviperceptorsmust be present close to the vascular cambium along the lengthof young woody stems. There is no reason to suppose that theperception of gravitational stimulus in stems is in any materialway different from that in roots.     

Wheat Callus Culture: the Initiation, Growth and Organogenesis of Callus Derived from Various Explant Sources

Callus was obtained from mature excised embryos of wheat, fromnodal and internodal stem segments and from rachis segmentsusing the medium of Murashige and Skoog(1962)(M medium), containing1-0mg l^–1 2,4-D, and from immature embryos using the mediumof Green and Phillips (1975) containing 2 mg l^–1 2,4-D.Callus yield from mature embryos depended upon the cultivarused. No callus could be obtained from leaf segments. Callusderived from mature embryos and nodal stem segments was successfullymaintained by serial sub-culture on the M medium containing2,4-D for up to 3 years although its growth rate declined toa lower level as culture proceeded. Such cultures consistently produced roots when transferred toa medium containing a low level of 2,4-D or no 2,4-D. The presenceof the auxin was essential for continued proliferation of thecallus tissue. Shoot initiation was infrequent, did not occurafter the first few sub-cultures and could not be enhanced byvarious auxin and cytokinin additions to the medium. Callusderived from immature embryos did not have an enhanced potentialfor shoot initiation. Triticum aestivum, wheat, callus culture, organogenesis     

Somatic Embryogenesis and Plant Regeneration from Freely-suspended Cells and Cell Groups of Panicum maximum Jacq

Embryogenic calluses derived from cultured immature embryosand young inflorescences of Panicum maximum Jacq. were placedin Murashige and Skoog's liquid medium supplemented with 1 mg1^–1 2, 4- dichlorophenoxyacetic acid (2, 4-D) and 2.5per cent coconut water, to initiate suspension cultures. Suspensionsconsisted of two types of cells: small, richly-cytoplasmic andoften starch-containing embryogenic cells, and large, vacuolatednon-embryogenic cells. A presumed sequence of developmentalstages from single embryogenic cells to globular and heart-shapedstages of embyrogenesis was observed in the suspension cultures.Plantlets were produced from the embryoids when the suspensionswere plated in an agar medium without any hormone or with only0.2 mg 1^–12, 4-D or naphthalene acetic acid. Embryogenicsuspension cultures derived from immature embryos as well asfrom inflorescence segments gave rise to plants which showedthe normal somatic chromosome number of 2n = 4x = 32. Panicum maximum Jacq., Guinea grass, embryogenesis, regeneration, suspension culture