rol-Transgenic Populus tremula: root development, root-borne bud regeneration and in vitro propagation efficiency

 The potential use of the rol genes from Agrobacterium rhizogenes to improve the root system horticultural characteristics was evaluated in transgenic aspen (Populus tremula) plants, harboring the rol genes under their native promoters. Southern blot and RT-PCR analyses confirmed the presence and expression of A. rhizogenes rolC and rolB genes in four different phenotypically selected transgenic clones. Several of the observed phenotypic modifications were related to rol-gene expression and included, in particular, modified root systems. All in vitro-cultured rol-transgenic plants exhibited extensive root formation in a hormone-free medium, as well as a larger root surface area and mass, as compared to a uidA (β-glucuronidase-encoding)-transgenic aspen line and control (non-transformed) plants. Adventitious root formation in stem segments of rol-transgenic plants exhibited very rapid kinetics, resulting in a much shorter rooting time for rol-transgenic stem segments (e.g. 10 days for 80% rooting in rol-transgenic lines T-26 and T-27, as compared to more than 18 days for control non-transformed or uidA-transgenic aspen plants). rol-Transgenic plants maintained the capacity for 100% rooting throughout the year, versus 70–80% rooting in non-transformed plants during the winter. The four rol-transgenic lines exhibited differences in root development; in two of them enhanced root development was accompanied by increased shoot fresh weight. The root:shoot fresh weight ratio was always higher in rol-transgenic lines than in non-transformed plants. In the T-27 rol-transgenic line, the propagation coefficient of shoot-bud regeneration in liquid root culture was almost three times higher than in non-transformed plants. To the best of our knowledge this is the first report on quantitative phenotypic alterations in rol-transgenic woody plants. Received: 1 December 1997 / Accepted: 8 March 1998     

Agrobacterium rhizogenes-mediated transformation of Taraxacum platycarpum and changes of morphological characters

Transformed hairy roots were efficiently induced from seedlings of Taraxacum platycarpum by infection with Agrobacterium rhizogenes 15834. Root explants produced transformed roots at a higher frequency (76.5±3.5%) as compared to stem (32.7±4.8%) or cotyledon (16.2±5.7%). Hairy roots exhibited active elongation with high branching of roots on growth regulator-free medium. The competence of plant regeneration from non-transformed adventitious roots and transformed hairy roots was compared. The frequency of adventitious shoot formation from transformed roots was much higher (88.5±9.8%) than that of non-transformed roots (31.7 ±9.5%) on hormone-free medium. Rooting of hairy root-derived adventitious shoots occurred easily on growth regulator-free medium but no rooting was observed on non-transformed shoots. The stable introduction of rol genes into Taraxacum plants was confirmed by PCR and Southern hybridization. Transgenic plantlets showed considerable differences in their morphology when compared to the corresponding wild-type (non-transgenic) plants. Plantlets formed from transformed roots had numerous fibrous roots with abundant lateral branches instead of the thickened taproots in non-transformed plants. The differences observed may reflect the modification of morphological root characters by introduction of rol genes.Communicated by M.R. Davey     

Wood formation in rolC transgenic aspen trees

Wood formation and structure of 3-year-old 35S-rolC transgenic aspen (Populus tremula L. × tremuloides Michx.) were compared with the situation in non-transformed control aspen trees. The transgenics are characterized by reduced shoot growth and an earlier bud break. Their wood formation did not immediately follow bud break and leaf development but occurred after some delay, about the same time as wood formation was initiated in the control trees. Quantitatively, the wood structure of the transgenics and of the non-transformed controls was not significantly different; therefore the dwarfism of the transgenics is very likely due to a reduction in cell number. Atypical formation of latewood led to the assumption that the differentiation of cells is decelerated. Additionally, cells lacked both secondary walls and normal lignification, and discoloration of the wood and the formation of tyloses were conspicuous in all transgenics. In contrast, they did not occur in the non-transformed control aspen trees. The observations are discussed in relation to the widely accepted auxin hypothesis. It is suggested that 35S-rolC transgenic aspen trees may be a useful model to study the regulatory mechanisms of wood formation. Received: 17 August 1999 / Accepted: 9 December 1999     

Trehalose 6‐phosphate is involved in triggering axillary bud outgrowth in garden pea (Pisum sativum L.)

Trehalose 6‐phosphate (Tre6P) is a signal of sucrose availability in plants, and has been implicated in the regulation of shoot branching by the abnormal branching phenotypes of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) mutants with altered Tre6P metabolism. Decapitation of garden pea (Pisum sativum) plants has been proposed to release the dormancy of axillary buds lower down the stem due to changes in sucrose supply, and we hypothesized that this response is mediated by Tre6P. Decapitation led to a rapid and sustained rise in Tre6P levels in axillary buds, coinciding with the onset of bud outgrowth. This response was suppressed by simultaneous defoliation that restricts the supply of sucrose to axillary buds in decapitated plants. Decapitation also led to a rise in amino acid levels in buds, but a fall in phosphoenolpyruvate and 2‐oxoglutarate. Supplying sucrose to stem node explants in vitro triggered a concentration‐dependent increase in the Tre6P content of the buds that was highly correlated with their rate of outgrowth. These data show that changes in bud Tre6P levels are correlated with initiation of bud outgrowth following decapitation, suggesting that Tre6P is involved in the release of bud dormancy by sucrose. Tre6P might also be linked to a reconfiguration of carbon and nitrogen metabolism to support the subsequent growth of the bud into a new shoot.     

In vitro plant regeneration in Holarrhena antidysenterica wall., through high-frequency axillary shoot prolieferation

Summary An efficient, rapid and large-scale propagation of the woody, aromatic and medicinal shrub, Holarrhena antidysenterica, through in vitro culture of nodal segments with axillary buds, is described. N⁶-benzyladenine used at 15 μM was the most effective in inducing bud break and growth, and also in initiating multiple shoot proliferation at the rate of 43 microshoots per nodal explant with axillary buds, after 30 d of eulture. By repeated subculturing of nodal explants with axillary buds, a high-frequency multiplication rate was established. Efficient rooting was achieved with 35 μM indole-3-butyric acid which was the most effective in inducing roots, as 80% of the microshoots produced roots. Plantlets went through a bardening phase in a controlled plant growth chamber, prior to ex vitro transfer Micropropagated plants established in garden soil were uniform and identical to donor plants with respect to growth characteristics and vegetative morphology.     

Gibberellins and Subapical Cell Divisions in Relation to Bud Set and Bud Break in Salix pentandra

In young plants of Salix pentandra, a temperate zone deciduous woody species, elongation growth ceases and a terminal bud is formed at day lengths shorter than a critical length. This is the first step in dormancy development, making survival under harsh winter conditions possible. Early studies strongly indicate that gibberellin is involved in the photoperiodic control of bud set and bud break. GA₁ action was studied by application under short days to plants where cessation of shoot elongation had occurred, followed by subsequent anatomic investigations of shoot tips. Under short days the frequency of cell division decreased rapidly along with the earlier observed decrease in GA₁ levels. Application of GA₁ to short-day–induced terminal buds rapidly stimulated cell division in apices several days before visible shoot elongation in response to this treatment was observed. One day after GA₁ application a fourfold increase in cell division frequency in apices was observed, increasing to a maximum of sevenfold 2 days after application. Long-day treatment leading to induction of bud break after about 4–6 days was followed by slowly increasing frequency of cell divisions. In earlier studies of this species, short days and gibberellins had no effect on cell elongation. These data show that increased GA₁ content, by application or long-day treatment, results in increased frequency of mitosis. This strongly indicates that GA₁ affects stem elongation in connection with bud set and bud break primarily by affecting cell divisions in subapical tissues. Received February 26, 1999; accepted October 8, 1999     

In vitro clonal propagation of an aromatic medicinal herb Ocimum basilicum L. (sweet basil) by axillary shoot proliferation

Summary An efficient protocol for in vitro propagation of an aromatic and medicinal herb Ocimum basilicum L. (sweet basil) through axillary shoot proliferation from nodal explants, collected from field-grown plants, is described. High frequency bud break and maximum number of axillary shoot formation was induced in the nodal explants on Murashige and Skoog (1962) medium (MS) containing N⁶-benzyladenine (BA). The nodal explants required the presence of BA at a higher concentration (1.0 mg·l⁻¹, 4.4 μM) at the initial stage of bud break; however, further growth and proliferation required transfer to a medium containing BA at a relatively low concentration (0.25 mg·gl⁻¹, 1.1 μM). Gibberellic (GA₃) at 0.4 mg·l⁻¹ (1.2 μM) added to the medium along with BA (1.0 mg·l⁻¹, 4.4 μM) markedly enhanced the frequency of bud break. The shoot clumps that were maintained on the proliferating medium for longer durations, developed inflorescences and flowered in vitro. The shoots formed in vitro were rooted on half-strength MS supplemented with 1.0 mg·l⁻¹ (5.0 μM) indole-3-butyric acid (IBA). Rooted plantlets were successfully acclimated in vermi-compost inside a growth chamber and eventually established in soil. All regenerated plants were identical to the donor plants with respect to vegetative and floral morphology.     

Growth Regulators in Populus tremula IV. Apical Dominance and Suckering in Young Plants

Experiments with small plants of Populus tremula L. growing in solution culture indicate that polarly transported auxin is an important factor in the control of axillary bud growth. If the auxin supply from the growing apex is eliminated, the number of buds released is influenced by factors translocated in the transpiration stream from the roots. Suckers may be induced to develop from aspen roots, the age of which is six weeks or more. Removal of the growing apex and the axillary buds or stoppage of shoot growth by short day treatment were effective in inducing abundant suckering in small aspen plants. Some mature leaves had to be maintained, indicating the dependence of sucker formation on carbohydrate supply. These treatments are known to decrease auxin production in the shoots. Extraction and biological assay showed a decrease in the content of auxin in the roots as a consequence of removal of growing shoot parts. The results indicate that suckering in roots of intact aspen plants is prevented by auxin transported into the roots from growing shoot parts.     

Shading delays bud break in Brachsyegia spiciformis

Whole tree manipulation experiments were performed in the common southern African tree species, Brachystegia spiciformis to test a novel hypothesis that decreasing Total nonstructural carbohydrates (TNC) in the stem could cause bud break in Brachystegia spiciformis. The experimental treatments included fertilization, canopy defoliation, shading and stem heating to decrease stem carbohydrates. None of the treatments significantly decreased mean stem TNC. Likewise the heating, fertilization and defoliation treatments did not significantly affect the date of bud break. However, shading significantly delayed bud break. This delay in bud break could not be attributed to changes in leaf level photosynthetic traits, stem water content, leaf predawn water potential or delayed leaf fall. These results question widely accepted hypotheses about the mechanisms controlling bud break and suggest a carbohydrate homeostatic mechanism.     

Gibberellic acid promotes in vitro regeneration and shoot multiplication in <Emphasis Type="Italic">Lotus</Emphasis><Emphasis Type="Italic">corniculatus</Emphasis> L.

Shoots of Lotus corniculatus L., previously transformed with Agrobacterium tumefaciens LBA4404/pTOK233, were grown in gibberellic acid (GA₃)-containing media in an attempt to improve their growth and multiplication. Nodal stem segments of four poorly multiplying clones, a clone with very good multiplication, and two non-transformed clones were incubated in media containing GA₃ for 3 weeks, and then returned to a hormone-free medium for a further 3–5 subculture periods. Gibberellic acid increased the number of axillary buds from one in controls to 2–13 shoots during the first 3 weeks of subculture in GA₃-containing media. The multiplied buds, which apparently derived from additional axillaries, continued to multiply in hormone-free medium, producing bunches of more than 50 shoots after 9 weeks. In addition, many nodal segments also produced callus tissues at the basal end, or along the internode below the node, in which abundant shoot meristems formed de novo. Histological examination confirmed their origin via organogenesis. About 25% of regenerated shoots rooted spontaneously. The application of auxins significantly improved rooting in more than 75% of all clones. Well-developed plantlets were produced upon the transfer of rooted shoots to pots. No differences in responses to hormones were observed between LBA4404/pTOK233-transformed clones and non-transformed clones, indicating that the reaction to GA₃ was not the consequence of transformation events. The results point to a possible involvement of GA₃ in lateral branching. They might also be recommended as a procedure suitable for increasing the production of transformed plants in L. corniculatus.     

Clonal micropropagation of <Emphasis Type="Italic">Crataeva adansonii</Emphasis> (DC.) Prodr.: A multipurpose tree

Summary A method of micropropagation through multiple shoot formation from axillary buds of mature tree and rootstock growths of Crataeva adansonii (DC.) Prodr. (a multipurpose tree) has been developed. Factors affecting multiplication rate included season, age of explant source, explant type, type of bud, position of bud on the foliage twig, type of medium, various additives, and explant implantation on the medium. The maximum number of buds was produced from the sixth to 10th axillary buds taken from foliage twigs of 40–50-d-old rootstock growth in the months of October to December. At this time of the year the contamination was minimum. Optimum response was recorded on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 3 mgl⁻¹, 13.3μM) and 1-naphthaleneacetic acid (NAA; 0.05 mgl⁻¹, 0.27 μM) after 21 d of culture. Shoot buds were further multiplied and maintained on BA (1–1.5 mgl⁻¹, 4.4–6.7 μM) while shoots elongated on BA (0.1–0.5 mgl⁻¹, 0.44–2.2 μM) supplemented medium. The number of shoots was further multiplied by using nodal segments of in vitro-regenerated shoots as microcuttings and repeated subculturing of stumps after excising the microshoots. In vitro rooting on growth regulator-free MS medium was possible with 70% of microshoots after 4 wk. From one nodal segment 150 plantlets were produced within 14 wk.     

Effect of plant growth substances on the growth of axillary buds in cultured stem segments of Phaseolus vulgaris L.

The hormonal control of axillary bud growth was investigated in cultured stem segments of Phaseolus vulgaris L. When the stem explants were excised and implanted with their apical end in a solid nutrient medium, outgrowth of the axillary buds-located at the midline of the segment-was induced. However, if indoleacetic acid (IAA) or naphthaleneacetic acid (NAA) was included in the medium, bud growth was inhibited. The exposure of the apical end to IAA also caused bud abscission and prevented the appearance of new lateral buds.In contrast to apically inserted segments, those implanted in the control medium with their basal end showed much less bud growth. In these segments, the auxin added to the medium either had no effect or caused a slight stimulation of bud growth.The IAA transport inhibitor N-1-naphthylphthalamic acid (NPA) relieved bud growth inhibition by IAA. This suggests that the effect of IAA applied at the apical end requires the transport of IAA itself rather than a second factor. With the apical end of the segment inserted into the IAA-containing medium, simultaneous basal application of IAA relieved to some extent the inhibitory effect of the apical IAA treatment. These results, together with data presented in a related article [Lim R and Tamas I (1989) Plant Growth Regul 8: 151–164], show that the polarity of IAA transport is a critical factor in the control of axillary bud growth.Of the IAA conjugates tested for their effect on axillary bud growth, indoleacetyl alanine, indoleacetic acid ethyl ester, indoleacetyl-myo-inositol and indoleacetyl glucopyranose were strongly inhibitory when they were applied to the apical end of the stem explants. There was a modest reduction of growth by indoleacetyl glycine and indoleacetyl phenylalanine. Indoleacetyl aspartic acid and indoleglyoxylic acid had no effect.In addition to IAA and its conjugates, a number of other plant growth substances also affected axillary bud growth when applied to the apical end of stem segments. Myo-inositol caused some increase in the rate of growth, but it slightly enhanced the inhibitory effect of IAA when the two substances were added together. Gibberellic acid (GA₃) caused some stimulation of bud growth when the explants were from younger, rather than older plants. The presence of abscisic acid (ABA) in the medium had no effect on axillary bud growth. Both kinetin and zeatin caused some inhibition of axillary buds from younger plants but had the opposite effect on buds from older ones. Kinetin also enhanced the inhibitory effect of IAA when the two were applied together.In conclusion, axillary buds of cultured stem segments showed great sensitivity to auxins and certain other substances. Their growth responded to polarity effects and the interaction among different substances. Therefore, the use of cultured stem segments seems to offer a convenient, sensitive and versatile test system for the study of axillary bud growth regulation.     

Phenotypic Effects of Isolated pRiA4 TL-DNA rol Genes in the Presence of Intact TR-DNA in Transgenic Plants of Solanum dulcamara L.

The effect of the rol genes, together with the TR-DNA of pRiA4on the phenotype of Solanum dulcamara plants, was analysed.Plants transformed by Agrobacterium strain BN1010: :rolA (rolA^7plus;TR⁺)exhibited severe leaf wrinkling, whereas plants transformedby strain BN1010: :rolC (ro/C⁺TR⁺) had a typical ‘hairyroot’ phenotype. Leaf discs excised from these latterplants produced roots on hormone-free medium. BN1010: :rolABC(rolABC⁺TR⁺) transformed plants had an exaggerated transformedphenotype. Some of the BN1010: :rolABC transformants had positivelygeotropic root growth which correlated with the presence ofmultiple copies of the TR-DNA. S. dulcamara plants, transformedby the TR-DNA region only, exhibited epinasty. Scanning electronmicroscopy of plants containing various regions of agropineRi T-DNA revealed that transformation causes changes in basicplant siructure.     

Expression of the <Emphasis Type="Italic">rol</Emphasis>B gene enhances adventitious root formation in hardwood cuttings of aspen

Summary An elite aspen hybrid (Populus × canescens × P. grandidentata) was transformed with Agrobacterium tumefaciens strain EHA105 that harbored a binary vector (pBI121) carrying the nptII gene under the nos promoter and tandem rolB-uidA (GUS) genes with the CaMV 35S or heat shock promoter. Among 32 independent kanamycin-resistant plants, 25 plants were confirmed by polymerase chain reaction and Southern blot analyses to contain all three genes, whereas five plants contained only nptII or/and uidA genes and two plants had both the rolB and nptII or uidA genes. Integration of the rolB gene significantly increased rooting ability of hardwood cuttings. Heat shock-rolB-transformed plants rooted at significantly higher percentage than the CaMV 35S-rolB-transformed plants. Heat shock treatment further enhanced rooting of heat shock-rolB-transformed plants. Exposure to exogenous auxin did not significantly increase the rooting percentage of transgenic hardwood cuttings, but increased the number of roots induced. This research shows great potential to improve rooting of hardwood cuttings of difficult-to-root woody plants which are commercially important to the horticultural and forestry industry. The transgenic plants with gain-of-function in hardwood-cutting rooting can facilitate research in the understanding of adventitious rooting from hardwood cuttings of recalcitrant woody plants.     

Nondormant mutants in a temperate tree species,Corylus avellana L.

Summary Nondormant mutants in hazelnut (Corylus avellana L.) are described. In contrast to normal trees in which physiological rest, or dormancy, is induced by short days, mutants fail to respond to this stimulus. Shoot tips continue to grow, old leaves are retained until midwinter when they are frozen and/or pushed off by developing axillary buds, axillary buds begin to grow in December, 2–3 months before normal spring bud break, and cold hardiness does not develop. Nondormancy is controlled by a single recessive gene (dd). The mutation is not uncommon since eight cultivars, including the world's most important commercial cultivars, are heterozygous for this trait. The implications of nondormancy in a temperate tree species are discussed in relation to evolution, extension of the range of cultivation, breeding, and value for basic studies of fundamental mechanisms of dormancy.     

Topophysis affects the Potential of Axillary Bud Growth, Fresh Biomass Accumulation and Specific Fresh Weight in Single-stem Roses (Rosa hybrida L.)

Topophysis, the effect on growth and differentiation of positionof axillary buds along the shoot, was studied by propagatingfive-leaflet-leaf single-node cuttings which were excised fromseven stem positions and grown as single stemmed plants. InRosahybrida ‘Korokis’ Kiss®, ‘Tanettahn’Manhattan Blue®, and ‘Sweet Promise’ Sonia®,following release of the buds from apical dominance by excision,morphogenetic development was studied until anthesis. The timefrom excision/planting until onset of bud growth, visible flowerbud appearance, and anthesis was generally shorter in plantsoriginating from apical bud positions than from basipetal positions.Topophysis mainly affected the onset of axillary bud growth;the earliest growth and development was found in cuttings fromthe second uppermost node position. This node tended to havethe lowest plastochron value, which indicated the existenceof a transition between sylleptic and proleptic buds. Stem lengthat visible flower bud and at anthesis generally increased asthe cutting position changed basipetally until the second lowestposition, and the number of five-leaflet-leaves at anthesisand the total number of nodes generally increased basipetally.For internode length, growth rate, and fresh biomass efficiencythe cuttings taken from the uppermost and lowermost positionsgenerally had significantly lower values than cuttings fromall medial positions. At anthesis, plants originating from cuttingsexcised from lower medial positions generally had a higher freshweight, greater flower stem diameter, and a significantly higherspecific fresh weight than those plants originating from apicalor basal positions. Among the cultivars, Sonia was the mostefficient in increasing fresh biomass and had the highest growthrate, whereas Manhattan Blue possessed the highest specificfresh weight, indicating a higher plant quality. It is suggestedthat topophysis inRosa is an independent phenomenon intrinsicto the axillary bud. apical dominance; axillary bud growth; fresh biomass accumulation; cut rose; flowering; Rosaceae; Rosa hybrida L.; rose; shoot growth; single-stem roses; specific fresh weight; topophysis; quality     

Stimulation of scion bud release by rol gene transformed rootstocks of Rosa hybrida L

For improvement of the root characteristics of roses, the rolA, B and C genes from Agrobacterium rhizogenes were introduced in the cultivar Moneyway, which is used as a rootstock. Four independent rolA+B+C transformants were produced. One rolC expressing transformant, showing good growth and clearly decreased apical dominance, was selected for a grafting experiment with the cut rose cultivar Madelon as a scion. Grafting on this rolA+B+C transformed rootstock resulted in a stimulation of both root development of the rootstock and axillary bud release of the untransformed scion. Axillary bud release increased from 0.1 to 0.6 and from 0.3 to 1.3 basal shoots per plant at 15C and 20C, respectively. Since basal shoots from flowers, this altered plant architecture will presumably lead to a more efficient flower production.Keywords: Agrobacterium transformation, apical dominance, rol genes, rootstock, rose.      

Micropropagation of chinese redbud (<Emphasis Type="Italic">Cercis yunnanensis</Emphasis>) through axillary bud breaking and induction of adventitious shoots from leaf pieces

Summary Factors affecting in vitro shoot production and regeneration of Cercis yunnanensis Hu et Cheng were investigated by comparing various growth regulators and explant types. For optimum shoot production from axillary buds, Murashige and Skoog (MS) media containing 6-benzyladenine, either alone or in combination with a low concentration of thidiazuron, resulted in the greatest number of shoots formed per explant (>3). Explants (2 mm long) containing one axillary bud placed in directcontact with the medium yielded the most shoots per bud (1.6) when grown on growth regulator-free medium. Root formation on 70–80% of shoot explants was accomplished using either indole-3-butyric acid or α-naphthaleneacetic acid in the medium, with significantly more roots formed on explants possessing and apical bud than those without the bud. Direct shoot organogenesis from leaf explants occurred on MS medium containing 10–30 μM thidiazuron, with up to 42% of leaf explants producing shoots.     

Plant growth regulator and graft control of axillary bud formation and development in the TO-2 mutant tomato

The torosa-2 tomato mutant is characterized by a strong inhibition of release of axillary shoots, that is not under the control of the main apex and IAA. Microscopic examination indicated that about 70% of leaf axils do not have axillary buds. Of the growth regulators tested, gibberellic acid and cytokinins were able to modify the to-2 phenotype: increasing bud number (GA₃ treated) and developing shoots (both substances). Sequential application of growth regulators demonstrated that bud production was only affected by treatments given between sowing time and 32 days after germination. Grafting experiments indicated that endogenous root factors have no essential role in the lateral branching of the genotypes investigated. The control of axillary bud differentiation and the branching pattern in the to-2 appears to be dependent of a complex mechanism involving gibberellins and cytokinins.     

The role of gibberellins and cytokinins in the growth correlative effect of cotyledons in flax and pea

It is wellknown that following the amputation, or darkening of one cotyledon in decapitated flax seedlings, the opposite remaining, or illuminated, cotyledon exerts a stimulatory effect on the growth of its axillary bud. For the induction of this stimulating effect a 21–72 h continuous darkening of the cotyledon is sufficient. Endogenous gibberellins take part in the stimulation effect of the illuminated cotyledon, since their level in the illuminated cotyledon increases as early as 12–48 h following the darkening of the opposite cotyledon. The apical part of the cotyledon has a higher growth stimulatory effect on the growth of the cotyledonary axillary bud than the basal half. This again is associated with endogenous gibberellins the level of which is higher in the apical half of the cotyledon than in the basal one. Upon removal of the root and hypocotyl base in decapitated flax seedlings deprived of one cotyledon, the remaining cotyledon loses its stimulatory influence, so that the bud of the amputated cotyledon grows more vigorously (Dostál 1955). In this growth correlative phenomenon the root may be substituted by cytokinin BA applied in the form of a 0.1–1.0 per cent paste onto the remaining cotyledon, for again in this case the bud of the preserved cotyledon grows more vigorously. Following the decapitation of the axillary of the amputated cotyledon in decapitated pea seedlings with an intact root and deprived of one cotyledon, the axillary of the remaining cotyledon grows more intensively than the serial of the removed one. If the plants operated on in the same way are deprived of the root, the serial of the removed cotyledon gains a correlative growth predominance. If the plants deprived of root are cultivated at the same time in a solution of BA (10–20 mg 1⁻¹), the correlative predominance is acquired by the axillary of the remaining cotyledon. In growth correlations between cotyledons and their axillary buds in pea seedlings the root may thus be substituted by exogenous cytokinin, as well.