Adventitious shoot regeneration from vegetative shoot apices in pear and putative role of cytokinin accumulation in the morphogenetic process

Adventitious shoot regeneration was obtained from callus produced from main vegetative apices of pear of in vitrogrown shoots of Italian cultivars Spadona and Precoce di Fiorano and wild pear genotypes ISF54 and ISF61. The highest morphogenetic response was obtained on a medium containing 8.8 M 6-benzyladenine, 1.0 M -naphthaleneacetic acid and 250 mg l^–1cefotaxime. The explants were maintained for 30 days in darkness and then transferred to an auxin-free medium and to the light. Histological studies revealed that the new vegetative buds originated from callus that completely altered the morphology of the explant tissues by the 30th day of culture. The in situ localisation of cytokinins, performed using antibodies with marked specificity against zeatin (Z) and isopentenyladenine, revealed an accumulation of Z in the cambiform cells of the leaf primordia and in the shell zone of the new forming buds showing a primary role of this cytokinin in cell differentiation of in vitro pear organogenesis.     

Change in shoot proliferation with repeated in vitro subculture of shoots of woody species of Rosaceae

Shoots of 6 ornamental species and cultivars of Rosaceae were repeatedly subcultured in vitro for 9 generations on Linsmaier and Skoog (1965) medium with the addition of BA. Shoot proliferation increased over the first few generations and then gradually declined in all species and at all BA concentrations tested with the exception of Chaenomeles japonica in which a decline in shoot formation occurred only at 5.0 mg 1^-1 BA. A decrease in shoot length and leaf size and an increase in the incidence of callus formation was observed after several subcultures. This apparently irreversible decline could be due to either genetic or epigenetic change resulting from repeated fluxes in cytokinin, nutrient status or sucrose, or to elimination of seasonal environmental fluctuations.     

Effects of agar concentration and vessel closure on the organogenesis and hyperhydricity of adventitious carnation shoots

Carnation plantlets (Dianthus caryophyllus L.) cultured in vitro often develop morphological and physiological anomalies, a phenomenon called hyperhydricity, which impairs their survival ex vitro. When the agar concentration of the growth medium was increased (from 0 to 12 g dm⁻³), thereby reducing water availability, the hyperhydricity of those adventitious shoots regenerated from carnation petals decreased. This was accompanied by a progressive fall in the water content of shoots (94.9 to 91.4 %), fresh mass (from 57.2 to 1.8 mg), number of leaf parenchyma cell layers (from 9.3 to 7.7), and the size of these cells (from 968 to 254 μm²). However, the number of regenerated shoots also decreased (17.7 in 2 g dm⁻³ agar to 4.3 in 12 g dm⁻³). Similarly, in ventilated tubes, which exhibit a lower relative humidity than tightly closed tubes, shoot organogenesis diminished up to 28 %, in tandem with shoot water content. Thus, relative humidity and water availability in culture vessels do not only influence shoot hyperhydricity in carnations, but also greatly affect adventitious shoot organogenesis.     

The effect of shoot and root genotype on phosphorus concentrations of shoots and roots

Genotypes of two morphologically different populations of white clover (Trifolium repens L.) were reciprocally and self-grafted. Ungrafted stolon tips were also grown as controls. Grafting per se had no significant effect on shoot size, root size, leaflet width or shoot and root % P. The scion genotype had a significant effect on shoot and root size, and leaflet width. Neither scion nor rootstock genotype had a significant effect on either shoot or root % P. However, there was a significant scion × rootstock × P level interaction for shoot % P. This along with other evidence suggests that conflicting results regarding effects of scion and rootstock on % P content of plants within species is probaby due to the interaction of scion and rootstock with environment.This work was undertaken at the Department of Agricultural Botany, University of Reading, Reading, England.This work was undertaken at the Department of Agricultural Botany, University of Reading, Reading, England.     

Changes in nutrient composition and pH of the culture medium during in vitro shoot proliferation of crabapple and pear

Immature soybean (Glycine max (L.) Merr) embryos, or cotyledons isolated from them, were cultured on modified MS medium containing B5 vitamins and NAA (50 M) to induce somatic embryogenesis. The effects of media variables, dissection treatments and light conditions were investigated in this system. The efficiency of embryogenesis increased as sugar concentration decreased from 12 to 1.5%; sucrose and glucose were similarly effective as carbon sources. In an examination of the effects of medium pH, values between pH 5.0 and 7.0 gave similar embryogenesis efficiencies, but the frequency of normal embryos was greater in media with low pH values. In buffered medium (10 mM MES), a pH of 5.0 was inhibitory to embryogenesis, and most normal embryos were produced at pH 5.5. Under various dissection treatments, embryogenesis efficiency and root and callus production were increased by tissue damage. Somatic embryogenesis was observed both in darkness and in light, although embryo development was impaired under high light (80 E m^-2 s^-1) conditions. The ability of somatic embryos to germinate was closely correlated with embryo normality, and was influenced little by the hormone content of germination media. Of various media tested for their ability to support the growth of germinated embryos, a medium based on hydroponic nutrient salts, supplemented with yeast extract, and gelled with Difco-Bacto agar gave the best plantlet growth.Abbreviations E m^-2 s^-1 microEinsteins per square meter per second - NAA -napthalene acetic acid - N50 MS salts with B5 vitamins and 50 M NAA (Napthalene acetic acid) - MES 2(n-morpholino) ethanesulphuric acid - BAP benzylamino purine - IBA indole butyric acid This paper (No. 86-3-97) is published with the approval of the director of the Kentucky Agricultural Experiment Station.     

Differential cytokinin effects on the stimulation of in vitro shoot proliferation from meristematic explants of castor (Ricinus communis L.)

A highly efficient and reproducible method of in vitro propagation using meristematic explants has been developed for castor. Embryo axes and shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 0.5–10.0 mg/l of adenine, N⁶-benzyladenine (BA), kinetin (Kn), thiadiazuron (TDZ) and zeatin. TDZ (1.0–10.0 mg/l) gave the maximum number of shoots (37.8–40.0) from embryo axes, while BA (2.0 mg/l) was found superior to other cytokinins for obtaining the highest number of shoots (46.7) from the shoot apex. Adenine and Kn at all of the tested concentrations resulted in low proliferation rates from embryo axes. The carryover effect of the cytokinins was tested by subculturing proliferating shoot cultures from various media onto the medium fortified with 0.5 mg/l BA. There was no significant influence of the cytokinins on subsequent proliferation from the two explant types except for TDZ with embryo axes. The number of shoots from TDZ-habituated embryo axes ranged between 36.0 and 81.7, while it varied from 5.7 to 22.0 and 3.7 to 28.3 in axillary buds and embryo axes, respectively, on the other media. For elongation of shoots, gibberellic acid (GA₃) (0.1–1.0 mg/l) was added to the medium supplemented with 0.2–0.5 mg/l BA. Incorporation of GA₃ (0.1 mg/l) significantly enhanced the frequency of elongated shoots but drastically reduced the multiplication ability. Hence, proliferating shoot clusters were periodically transferred to the medium supplemented with 0.5 and 0.2 mg/l BA for further multiplication and elongation. Well-developed shoots were rooted on half-strength MS medium supplemented with 1.0 mg/l indole-3-butyric acid. The rooted plantlets were acclimatized with more than 60% success. Received: 17 June 1997 / Revision received: 3 September 1997 / Accepted: 20 September 1997     

In vitro phyllomorphic regeneration of shoot buds and shoots in Picea abies

Needles (10–15 mm) of frost-hardened 20–22-week-old (physiological age equivalent to 1 year) plants of Picea abies L. excised just after flushing, were induced to form adventitious shoot buds and shoots on media supplemented with BAP (6-benzylaminopurine) and NAA (1-napthaleneacetic acid). The addition of nanomolar concentrations (0.5–50) of NAA combined with 1–10 μM BAP considerably stimulated formation of pseudobulbils on the basal to mid-part of the needle axis, as well as their subsequent development into shoot buds and shoots. On a medium containing 10 μM BAP, pseudobulbils that formed at the needle base did not develop further, but became necrotic and died with the omission of NAA. With 5 μM BAP + 50 nM NAA the initial phase of development was slow, but later showed good response and up to 22% of the needles produced shoot buds. Two to three shoots per needle could be excised and subcultured individually onto fresh media. It is concluded that the level of endogenous auxin decreases progressively from the needle's base to its tip, so that that concentration of exogenous auxin (50 nM NAA) which promotes pseudobulbil and shoot-bud formation part-way along the needle axis, simultaneously inhibits their induction at the needle base.     

Effect of CPPU on in vitro axillary shoot proliferation and adventitious shoot regeneration in kiwifruit

Abstract

The effect of CPPU, N‐(2‐chloro‐4‐pyridyl)‐N‐phenylurea, on the development of axillary buds and on adventitious shoot regeneration was investigated in mature leaves of in vitro‐cultured shoots of Actinidia chinensis Planch (Sel. K190) and Actinidia deliciosa A. Chev. cultivars Hayward and Tomuri. In the multiplication phase, 2.2 mM CPPU induced proliferation rates comparable to 4.4 mM benzyadenine (BA) both in Hayward and in Tomuri, while a higher CPPU concentration reduced proliferation. In A. chinensis, significant differences in multiplication rates were not detected between BA and CPPU. However, shoots developed on CPPU appeared hyperhydric, and had very short internodes, reduced leaf laminas, higher water, carotenoid and phenol contents and considerably lower chlorophyll level in comparison with the BA‐grown shoots. On the other hand, in adventitious shoot regeneration CPPU was more effective than zeatin (ZEA) and BA in A. deliciousa cultivars and the best morphogenic response was obtained with the lowest concentration (10 mM) in cv Hayward, while 16 mM was the most efficient treatment in cv Tomuri. In A. chinensis, CPPU was as efficient as ZEA when the highest concentration was used.     

Effects of source of macronutrients and plant growth regulator concentrations on shoot proliferation of Cornus florida

Nodal stem segments of flowering dogwood (Cornus florida L.) were cultured on media containing seven different sources of macronutrients including full- and half-strength Murashige & Skoog (MS) macrosalts, N6, Anderson's (AND), Quoirin & Lepoivre's (LP), Nitsch & Nitsch (NIT), and Woody Plant Medium (WPM). All media contained MS micronutrients, Staba vitamins, 20 g l^-1 sucrose, and 6.5 g l^-1 Difco Bacto-agar, and were supplemented with 2.2 M 6-benzyladenine (BA) and 0.49 M indole-3-butyric acid (IBA). Following three subcultures, the best shoot proliferation was supported on media containing WPM macronutrients. To optimize the proliferation rate, shoots were cultured on WPM macronutrients supplemented with eight combinations of BA and IBA, and 3.3 M BA without IBA was determined to be the best.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid     

Effects of pear fruit-derived volatiles and their concentrations on the responses of oriental fruit moths

Grapholita molesta (Busck) is an important pest of pear trees. Numerous esters accumulate consistently in mature pear fruits. However, little is known about the effects of single esters from pear fruits at different concentrations on the responses of male and female G. molesta. In this study, the responses of virgin males, virgin females, and gravid females to five esters (3-methylbutyl acetate, ethyl hexanoate, ethyl butanoate, butyl acetate, and hexyl acetate) of pear fruits at three dosage levels (1, 5 and 10 μg/μL) were investigated using electroantennography (EAG), wind tunnel experiments, and field tests. Our results showed that ethyl butanoate (5 μg/μL) elicited higher EAG responses in virgin females and males than a mixture of all five volatiles, and elicited an equal EAG response to that for the mixture in gravid females, with all responses to ethyl butanoate and the mixture being higher than the response to hexane alone. For upwind flight, ethyl butanoate (5 μg/μL) excited equal moth flight–location behavior in virgin and gravid females to that for the mixture, which were both higher than the response to hexane. For close flight, ethyl hexanoate (5 μg/μL) and 3-methylbutyl acetate (10 μg/μL) appeared to be the activating compounds, eliciting equal responses from virgin females to that of the mixture, and higher responses of gravid females to that of the mixture, both of which were higher than the responses to hexane. Upwind and close flight responses of virgin males were lower than those of virgin and gravid females. In field trials, we verified that traps baited with ethyl butanoate (5 μg/μL) captured more moths than the other esters. Therefore, from the esters and concentrations tested, we recommend ethyl butanoate (5 μg/μL) for potential use as an attractant for G. molesta in orchards.     

Adventitions shoot formation on excised leaves of in vitro grown shoots of apple cultivars

Leaves taken from micropropagated shoots of several apple (Malus domestica Borkh.) cultivars were cultured in vitro on Linsmaier & Skoog (LS) medium or the rice anther culture medium of Chu et al. (N6) containing various concentrations of either benzyladenine (BA) or thidiazuron (TDZ) plus naphthaleneacetic acid (NAA). Of the TDZ concentrations tested, 10 M was most effective and it was equivalent to, or better than, 22 M BA for both the percentage of leaves regenerating shoots and number of shoots formed per regenerating leaf in almost every experiment. Lower concentrations of NAA (1.1 and 5.4 M) gave best results with both BA and TDZ. N6 medium gave consistently better results than LS. Lowering total salt concentration or total N concentration of LS to that of N6 did not improve the response nor did changing the NO₃:NH₄ ratio. The 3–4 leaves on the most distal part of the shoot were most responsive and tended to form the most adventitious shoots. Placing the leaf cultures in the dark for the first 2–3 weeks of the culture period produced the best results. Optimum results were obtained by culturing leaves from the distal part of the shoot in the dark for 2 weeks on N6 medium containing 10 M TDZ and 1.1 or 5.4 M NAA, then moving the cultures to 16 h daylight at a photon flux of 60 mol s^-1m^-2.     

In vitro shoot regeneration of Populus deltoides: effect of cytokinin and genotype

Treatment differences were observed in the in vitro adventitious shoot regeneration response from internodal explants of three genotypes of Populus deltoides cultured on media supplemented with five concentrations each of the cytokinins 6-benzyladenine, 2-isopentyladenine, and zeatin. For each of the three genotypes, the greatest number of shoots were consistently regenerated on media containing the cytokinin zeatin. Tissue necrosis resulted when explants from any of the three genotypes were cultured on media supplemented with 6-benzyladenine. A zeatin concentration by genotype interaction was also observed. Genotypic differences in shoot regeneration were observed for 16 genotypes of Populus deltoides when cultured on medium supplemented with 0.5 mgL^–1 zeatin. Six genotypes were highly recalcitrant and failed to regenerate shoots. The percent of explants regenerating was greater than 50% for four genotypes.Abbreviations WNA modified Woody Plant Media - BA N⁶-benzyladenine - 2-iP 2-isopentenyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAR photosynthetically active radiation Journal Series No. 8938, Agricultural Research Division, University of Nebraska     

In vitro proliferation of shoots and regeneration of cotton

Shoot proliferation from different explants of several Indian cultivars of cotton was studied in culture. Cotyledonary nodes taken along with the shoot apex of seedlings produced multiple shoots on modified MS nutrient agar supplemented with cytokinins. 6-Benzyladenine was most effective in inducing growth of multiple shoots. Explants of several genotypes formed organogenic masses that differentiated to secondary shoots on repeated subculture. The isolated shoots were rooted on basal medium supplemented with naphthaleneacetic acid and were transferred to soil after acclimatization. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors affecting in vitro shoot formation from vegetative shoot apices of apple and relationship between organogenic response and cytokinin localisation

ABSTRACT

The effects of macro- and micro-elements, benzyladenine (BA) concentration, and the period of auxin application on adventitious shoot formation from callus originating from vegetative shoot apices were tested on apple (Malus domestica Borkh) rootstock Jork 9. The putative relationship between organogenic response and cytokinin localisation was also studied by an immunolocalisation technique for in situ determination of free cytokinins. The use of MS (Murashige & Skoog, 1962) salts in the medium instead of those of LP (Quoirin & Lepoivre, 1977) had a strong positive effect both on shoot formation rate and on the number of shoots produced. The highest organogenic response from callus was induced using 17.8 μM BA in the presence of 2.7 μM NAA and by maintaining the explants for 20 days in darkness, then transferring them to fresh auxin-free medium and to the light. The in situ localisation studies, performed using antibodies with a marked specificity against zeatin and isopentenyladenine, revealed changes in the localisation of free zeatin in the tissues during the shoot-forming process, in particular during the active cell division phase leading to callus formation, and in the initial phase of bud formation. Changes in zeatin distribution in the tissues of the vegetative shoot apex during shoot formation may indicate a role for this cytokinin free base in cell differentiation and organogenesis.     

Factors affecting in vitro shoot proliferation and ex vitro establishment of mangosteen

Shoot proliferation has been achieved in Garcinia mangostana L. using seed explants. Maximum mean number of shoots per explant (16.8) was obtained from cultures on Murashige and Skoog medium supplemented with 40 mM 6- benzyladenine, and 2.5 mM -naphthaleneacetic acid and kept at 30 °C under an 8 hour photoperiod. Cultures on the same medium but supplemented with 2 g l^-1 activated charcoal produced fewer shoots. However, growth of these shoots was more organized and 75% rooting was obtained. Woody Plant Medium was not a suitable medium for shoot proliferation. Ex vitro establishment was best obtained on planting medium consisting of sand, soil and organic material (3:2:1).Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid - MS Murashige & Skoog (1962) basal medium - WPM Woody Plant basal medium (Lloyd & Mc Cown 1980)     

Endogenous cytokinins in shoots of Aloe polyphylla cultured in vitro in relation to hyperhydricity, exogenous cytokinins and gelling agents

The process of hyperhydricity in tissue cultured plants of Aloe polyphylla is affected by both applied cytokinins (CKs) and the type of gelling agent used to solidify the medium. Shoots were grown on media with agar or gelrite and supplemented with different concentrations of N⁶-benzyladenine (BA) or zeatin (0, 5 and 15 μM). Endogenous CKs were measured in in vitro regenerants after an 8-weeks cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. On media with agar a reduction in hyperhydricity occurred, while the gelrite treatment produced both normal and hyperhydric shoots (HS). The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of HS was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene.     

Control of hyperhydricity in Eucalyptus axillary shoot cultures grown in liquid medium

Shoots of four clones of two Eucalyptushybrids were successfully grown in a liquid medium containing modified MS basal salts supplemented with 0.01 mg l ^–1 (0.04 M) BAP and various concentrations of a proprietary anti-hyperhydricity agent (sold commercially as anti-vitrification agent EM2). Shoots of a single clone were also grown using the same medium but with the anti-hyperhydricity agent replaced with various concentrations of a commercially available gelling agent containing pectin (M-Gel) or a polysaccharide extract (iota-carrageenan) from seaweed. A range of supplements applied to the media were effective in reducing the hyperhydricity of shoots. The addition of EM2 and M-Gel led to a significant decrease in hyperhydricty, without showing any significant detrimental effect on the numbers of shoots produced for three of the four genotypes tested. At a concentration of 5 g l^–1, for either compound, the percentage rooting and percentage survival of shoots, both in vitro and ex vitro, either equalled or exceeded those of untreated shoots. Addition of iota-carrageenan, although beneficial in reducing hyperhydricity and improving percentage rooting in vitro, was detrimental to shoot production and acclimatisation ex vitro. Common factors of these agents' contributions to their anti-hyperhydric properties are discussed.     

Agar and ammonium nitrate influence hyperhydricity,tissue nitrate and total nitrogen content of serviceberry (Amelanchier arborea) shoots in vitro

Shoot tip cultures of Amelanchier arborea Michx.f. were grown on Murashige & Skoog or Woody Plant (WP) medium containing 4.4 M benzyladenine and various concentrations of agar. Increases in agar concentration affected various culture growth variables, decreased culture hyperhydricity and increased tissue nitrate concentration. Additions of ammonium nitrate to cultures grown on WP medium containing 0.4% agar increased all growth variables measured except percent dry weight. Hyperhydricity and tissue nitrate concentration also increase in response to increasing ammonium nitrate in the medium. Since hyperhydricity was shown to be both positively and negatively correlated with increases in tissue nitrate content, it is unlikely that tissue nitrate level alone directly affects hyperhydricity.Abbreviations BA benzyladenine - MS Murashige & Skoog - WP Woody Plant     

Phloroglucinol enhances growth and rate of axillary shoot proliferation in potato shoot tip cultures in vitro

Efficacy of phloroglucinol in promoting growth and development of in vitro-derived shoot tips was studied in six potato (Solanum tuberosum L.) genotypes. Different concentrations of phloroglucinol (0, 0.08, 0.4, 0.8, 1.2 and 1.6 mM) were tested in combination with either 0.1 or 0.2 M sucrose in shoot tip proliferation medium based on MS (Murashige and Skoog, 1962) medium supplemented with 5.8 μM GA₃ (gibberellic acid), 1.1 μM BA (N⁶-benzyladenine) and 8.39 μM D-calcium pantothenate. Phloroglucinol fostered multiple shoot formation, promoted axillary shoot proliferation in terms of shoot tip fresh weight and shoot length, and stimulated root formation on the shoot tips. There was significant phloroglucinol × sucrose interaction for number of shoots developed per shoot tip, shoot tip fresh weight and number of roots induced per shoot tip. The beneficial effect of phloroglucinol on shoot tip survival was conspicuous only in genotypes that showed poor survival in the control proliferation medium. There were significant differences in response between the two sucrose levels with regard to shoot tip fresh weight and number of roots per shoot tip. Phloroglucinol in combination with 0.2 M sucrose induced maximum number of roots per shoot tip. Optimum shoot tip growth was fostered in medium containing 0.8 mM phloroglucinol and 0.2 M sucrose. High frequency multiple shoot formation in this medium ensures a faster rate of potato shoot tip multiplication within a limited time and space. This revised version was published online in June 2006 with corrections to the Cover Date.