Adventitious Budding Research Articles

Micropropagation of Carob (<i>Ceratonia silique</i> L.) through Adventitious Buds of Immature Embryonic Cotyledons

Adventitious budding from embryonic cotyledons of immature seeds of carob was obtained. The combination of BAP (4.44 μM) and NAA (1.5 μM) furthered the neoformation of adventitious buds. These latter were multiplied on MS medium added with BAP (2.22 μM). Stems and leaves growing were improved by adding 2.02 μM GA3. Elongation was favored by 0.5 μM NAA. 70% of rooting was obtained with 10 μM IBA.

Flow-resistance adaptations of rigid-stemmed aquatic macrophytes in simulated water channels

This study investigated the development of interspecific adaptations of flow-resistance mechanisms to higher flow rates in rigid-stemmed Hygrophila salicifolia (Vahl) Nees (willow leaf Hygrophila sp.) plants placed in simulated water channels. The results indicate that adaptations to higher flow rates include a reduction in: growth rate, average fresh weight, average dry weight, and average diameter; but an increase in the number of parallel shoots. These effects combine to create a streamlined profile, reduce plant damage, and increase propagation through adventitious budding. Higher flow rates also reduced the ratio of average plant height to average root length in rigid-stemmed Hygrophila sp. The increased root length, strengthening of plant anchors, and reduction of uprooting seen at higher flow rates are likely to increase slope stability and reduce riverbank topsoil runoff. Moreover, rigid-stemmed aquatic macrophytes develop different adaptations than flexible-stemmed water plants (e.g., water celery); for these plants, higher flow velocities trigger an increase in the average density of vascular bundles and a reduction of the average root length, which results in uprooting and movement to different locations. These results suggest that different aquatic macrophytes play different roles in water channels. Our methods and findings can inform further investigations into the roles played by different aquatic macrophytes in ecological engineering and help to identify optimal planting materials or precursors for riverbanks.

In Vitro Micropropagation of Jatropha curcas L. from Bud Aggregates

Entire plants were regenerated from nodes explants of Jatropha curcas L. following a procedure of bud aggregate induction on MS (Murashige and Skoog) medium supplemented with 25 mg.l-1 citric acid, 12.2 mg.l-1 adenine sulfate, 15 mg.l-1 L-arginine, 2.46 µM IBA (indole-3-butyric acid), 30 g.l-1 sucrose and 7 g.l-1 of agar, and enriched with different balances of BA (benzyladenine) and L-glutamine. The histological studies performed on aggregates showed that the buds result from both the development of axillary buds and adventitious budding starting from underlying tissues of the explant. The culture medium containing 6.65 µM BA and 25 mg.l‑1 L-glutamine gave the best results with an average of 64 buds per aggregate after three weeks for all accessions tested. The buds developed into shoots when placed in an MS medium supplemented with 2.21 µM BA, 5.70 µM IAA (indole-3-acetic acid) and 15 mg.l-1 L‑arginine. These shoots were isolated and then rooted in MS containing 2.46 µM of IBA, 2% sucrose and 0.7% agar. The entire process took 13 weeks with a 98% survival rate in terms of plantlets acclimatization. We obtained a multiplication rate of 13 buds per explant and per subculture which is the double of those obtained in other recent works based on the micropropagation of J. curcas from node explants. This protocol is economically more profitable.

In vitro direct plant regeneration and Agrobacterium‐mediated transformation of lucerne (Medicago sativa L.)

AbstractIn vitro direct plant regeneration of lucerne was achieved by simultaneous application of thidiazuron (TDZ) and 6‐benzyladenine (BA) in Murashige and Skoog (MS) medium. Seedlings were germinated and grown for 6 d on growth regulator–containing MS medium. The shoot tip, consisting of the apical meristem along with parts of the cotyledonary leaves and hypocotyl, was then cultured on a medium containing the growth regulator(s). Adventitious budding of the shoot tip was promoted synergistically by treatment with TDZ and BA, and a maximum of thirty‐five shoots per explant was obtained on a medium supplemented with 2 mg L−1 TDZ and 1 mg L−1 BA. Plant regeneration frequency varied from 67 to 93%, and five Indian lucerne cultivars responded well to the regeneration protocol. The Agrobacterium‐mediated transformation frequency from co‐cultivated explants was 13% following multiple shoot induction. Southern analysis of the T0 plants and T1 progenies confirmed stable inheritance of the hpt marker gene. Agrobacterium infection of the explant caused a significant reduction in the plant regeneration frequency (23%) and the number of shoots induced (11) when compared with uninfected explants. A single shoot tip provided sufficient material to regenerate and establish twenty‐seven lucerne plants, whereas only nine plants could be regenerated from an Agrobacterium co‐cultivated explant. This transformation protocol could represent a valuable improvement over existing ones for lucerne.

Effects of the shoot-cutting method on field propagation in napiergrass (Pennisetum purpureum Schum.)

In this study, the effects of shoot-cutting method on propagation using overwintering napiergrass (Pennisetum purpureum Schum.) buds were examined under soil conditions compared with plant growth regulators (PGRs) that are used as an effective method for increasing adventitious buds of several gramineous crops. To increase adventitious budding, we cut shoots of napiergrass at 2 weeks after planting and treated with the following four types of PGR in comparison with shoot-cutting: 4.44 mM BA as cytokinin, 3.63 mM triazine as anticytokinin, 5.37 mM NAA as auxin, and 2.22 mM TIBA as antiauxin, either by injection or direct spraying onto the buds. A large number of adventitious buds were observed in BA- and TIBA-injected buds at 4 WAT compared to shoot-cut or PGR-sprayed buds. However, an extremely low survival rate for tiller-divided plants (nursery plants) derived from both BA- and TIBA-injected plants was observed compared with non-PGR controls. Similar trends on the survival rate and relative tiller number in main shoot-cut plants derived from intact buds (without PGR treatment) were also observed in a repeat trial. The present study demonstrated that the method of shoot-cutting could contribute to increasing propagation efficiency for nursery plants compared with exogenous PGR application, and would be a practical method for increased nursery propagation of napiergrass under soil conditions.

Morphology and ecology of the Antarcticycas plant

The Antarcticycas plant is reconstructed from the co-occurring vegetative organ taxa Antarcticycas schopfii and Yelchophyllum omegapetiolaris and the pollen cone taxon Delemaya spinulosa from permineralized peat of the Fremouw Formation, early Middle Triassic of Antarctica. The association of the organs in the same deposit, assignment of each taxon to Cycadales, and histological features shared among the taxa suggest they were all produced by the same plant. The Antarcticycas plant is reconstructed as a small plant, possibly with a subterranean stem; it was probably similar in habit to small-stemmed species of extant Zamia. Antarcticycas stems are inferred to exhibit apical rather than axillary branching based on reinterpretation of type material that shows a branching axis accompanied by anatomy indicative of the presence of cone domes when observed in extant cycads. The presence of a bulbil on one specimen indicates that branching also took place through adventitious budding. The Antarcticycas plant is similar in its anatomy to extant cycads, although contractile tissue and coralloid roots have yet to be identified and may not have been present in the fossil plant. The plant inhabited a warm temperate polar habitat with protracted periods of winter darkness for which there is no modern equivalent. Evidence for deciduousness in this taxon, previously suggested as a possible adaptation to its warm, light-limited environment, is equivocal. The possibilities that fire played a role in the Fremouw peat ecosystem and that the Antarcticycas plant may have been insect pollinated are explored.

ANEUPLOIDY, A SOURCE OF GENETIC DIVERSITY FOR FRUIT SPECIES

Aneuploidy is reported among several plant species. Issued from open-pollinated polyploid plants and from targeted breeding, most genotypes can be saved after embryo culture that prevents plant failure due to abortion and growth abnormalities respectively. Aneuploid apple material revealed genetically stable through in vitro axillary branching and crossable after ex vitro grafting. However, certain genetic variability occurred after adventitious budding, spontaneously inducing polyploidisation and cytochimerism. Some cytochimerical clones probably sectorial chimeras, reversed to genetically uniform aneuploids after subcultures by axillary branching. Multiapexing and adventitious budding applied to these chimeras led to the regeneration of new genotypes characterized by stable and uniform ploidy or aneuploidy and mixoploidy as well. Different ploidy patterns of regenerants resulted from the hormonal balance of the shoot induction medium. The combination of the aneuploidy with the techniques of in vitro regeneration led to create new genotypes with a limited genetic variability. These are currently used for genetic analysis and the production of seedless fruit. It appears obvious that with the use of specific molecular markers, this way of genetic improvement could support the progression of innovative breeding programs.

REGENERATION-PROMOTING GENES IMPROVE TRANSFORMATION EFFICIENCY IN APRICOT

Efficient selection of transformed plants is one of the limiting steps in the transformation of mature tissue of most woody fruit trees. The transformation with regeneration-promoting genes (i.e. ipt gene) may improve results in recalcitrant species. Transformation strategies with this gene include the use of inducible promoters or the removal of the ipt gene from the transformed plant. The later strategy is the base of the MAT vector system, a construction that include a recom-binase and specific sequences for the enzyme flanking the marker genes and the ipt. In this work, results from the utilisation of these constructions to transform apricot leaves are presented. Since ipt expression is supposed to induce regeneration of adventitious shoots in the absence of growth regulators, our standard transformation procedure was modified eliminating all growth regulators from the culture media. However, explants died in these conditions. When an auxin (NAA) was added to the regeneration medium at different concentrations, only production of adventitious roots was obtained from the transformed leaves. Therefore, addition of a cytokinin was necessary to avoid production of roots and to induce adventitious budding. The NAA concentration used in our standard procedure was combined with different TDZ concentrations during the co-culture period. These cytokinin concentrations were kept or lowered in the regeneration medium after co-culture. All regenerated buds were cultured in a medium specifically developed to grow apricot meristems into shoots that can be micropropagated. Kanamycin 60 μM was added to this medium to screen for those that were transformed. Transformation efficiencies obtained with the ipt gene have been three-fold those obtained with our standard transformation procedure and also selection efficiencies are much higher and, in some of the treatments with lower transformation efficiencies (3-5.7%), were as high as 50%. Moreover, the constructions used should allow elimination of marker genes and production of marker-free transformed apricot plants, which is the next aim of our research.

Factors influencing axillary shoot proliferation and adventitious budding in cedar

We developed procedures for in vitro cloning of Cedrus atlantica Manetti and C. libani A. Rich explants from juvenile and mature plants. Explant size was one determinant of the frequency of axillary bud break in both species. Shoot tips and nodal explants mainly developed calli, whereas bud sprouting occurred in defoliated microcuttings cultured on a modified Murashige and Skoog medium without growth regulators. Isolation and continuous subculture of sprouted buds on the same medium allowed cloning of microcuttings from C. atlantica and C. libani seedlings and bicentennial C. libani trees, thus providing a desirable alternative for multiplying mature trees that have demonstrated superior characteristics. We also report adventitious bud differentiation from isolated embryos of C. atlantica. Neither auxin treatments nor other methods tested, including infection with Agrobacterium rhizogenes, were effective in inducing root initiation.

Application of rapd in the determination of genetic fidelity in micropropagated Drosera plantlets

Random amplified polymorphic DNA (RAPD) markers were used to verify the clonal fidelity of two micropropagated Drosera species, D. anglica and D. binata, which were regenerated by adventitious budding from leaf explants and shoot tips, respectively. Twenty arbitrary decamers were used to screen 15 randomly selected plantlets of each species. No genetic variation was detected among D. binata regenerants, whereas a 0.08% polymorphism frequency was estimated for D. anglica plantlets. These results indicate that the regeneration of plants through shoot-tip culture is a low-risk method for generating genetic variability, whereas material regenerated through leaf explants requires further verification.

Gelling agent influences the detrimental effect of kanamycin on adventitious budding in flax

Flax (Linum usitatissimum L.) hypocotyls were cultivated on regeneration media containing various concentrations of kanamycin (an aminoglycoside antibiotic commonly used to select transgenic plant material) solidified with three different gelling agents: gellan gum, agar and a mixture of both. The inhibitory effect of kanamycin on bud regeneration was analyzed. A significant interaction was observed between the nature of the gelling agent and the kanamycin concentration. The antibiotic concentration needed to strongly inhibit bud production varied greatly with the nature of the gelling agent. Gellan gum lowered the inhibitory effect of kanamycin.

The detection of somaclonal variants of beet using RAPD

Plantlets were regenerated by adventitious shoot budding in tissue culture from leaf explants of a single genotype of sugar beet. DNA was extracted from the parental plant and from 120 regenerants. RAPD analysis was carried out using five decanucleotide primers; 4,557 RAPD marker bands were examined and two polymorphisms were observed. Thirty secondary regenerants were then derived, using the same tissue culture technique, from thirty of the primary regenerants. Again RAPD analysis was employed and a single band polymorphism was observed out of 1,050 bands examined. The overall frequency of detection of somaclonal polymorphisms using RAPD (3 in 5,607 = 0.05%) is similar to frequencies previously reported using isozyme and RFLP technologies.

Morphological features as indicators of maturity in acclimatized Pinuspinaster from different invitro origins

Acclimatized Pinuspinaster Ait. plants originating from five different invitro culture origins were compared morphologically. After one growing season in the greenhouse, the plants displayed the following order of maturity (from juvenile to mature): (i) young seedlings; (ii) plants produced by adventitious budding from cotyledons; (iii) plants derived from micrografted meristems of 2-year-old seedlings; (iv) plants derived from micrografted meristems of a 13-year-old mature clone; and (v) plants derived from microcuttings of axillary shoots of the same 13-year-old mature clone. Plants derived from micrografts exhibited substantial variability with regard to morphological characteristics. This variability included some striking demonstrations of morphological rejuvenation among the mature clone micrografts which, on average, showed more juvenile traits than the microcuttings of the same clone.

Direct organogenesis from internodal segments of in vitro grown shoots of apple cv. Golden delicious

A protocol for direct organogenesis from internodal segments of in vitro grown shoots obtained from mature apple cv. Golden delicious trees is presented. Adventitious buds were initiated on Murashige and Skoog medium (1962) containing various combinations of benzylaminopurine (BAP) and 2,3,5-triiodobenzoic acid (TIBA). Low concentration of BAP (4.4 μM) in combination with TIBA (1 μM) gave the best percentage of regeneration. Three repeated cycles of culture and regeneration produced an increase of adventitious budding up to 23%. Although no auxin was used in the organogenic medium, callus was always obtained. The regenerated shoots were micropropagated and rooted. Cytological studies revealed that proliferating buds originated directly from the superficial layers of the internodal explants without an intermediate callus phase.

Morphological, Histological and Lipid Changes during Adventitious Budding inPinus pinasterCultured Cotyledons

Tranvan, H., Troton, D. and Calvayrac, R. 1988. Morphological, histological and lipid changes during adventitious budding in Pinus pinaster cultured cotyledons.—J. exp. Bot. 39: 907–915. Isolated cotyledons of Pinus pinaster were cultured under bud-forming conditions. The morphology, histology, lipid content and the chlorophyll pools of the cotyledons were followed during bud development. Comparison with cotyledons cultured continuously on hormone-free or hormone-supplemented medium was performed. Three periods were distinguished during bud formation: (i) the initiation period, characterized by a drastic decrease of triacylglycerols, enhanced by the presence of the hormones; (ii) the differentiation period, during which the intense cell divisions and the increase of the polar lipid and chlorophyll contents appeared strongly correlated and (iii) bud development, characterized by stable levels of both chlorophylls and neutral lipids. Moreover, it appeared that the continuous presence of hormones in the culture medium inhibited the synthesis of molecular components, in particular chlorophylls and membrane lipids, required for normal cell differentiation and physiology.

Germplasm and Physiologic Effects on Induction of High‐Frequency Hormone Autonomous Callus and Subsequent Shoot Regeneration in Sugarbeet1

The availability of adapted germplasm capable of regenerating shoots from callus may affect the speed with which tissue culture‐mediated genetic improvements can be brought to commercial usage. To assess the shoot regeneration capability in Beta vulgaris L., 17 germplasm sources including sugarbeet, table beet, fodder beet and leaf beet were screened for callus‐forming ability and shoot regeneration. Petiole and blade explants from in vitro shoot cultures grown with 6‐benzyladenine (BA) as the only growth regulator were challenged to initiate callus on a Murashige‐Skoog basal medium (MS) with 0.25 or 1.0 mg L−1 BA. Bud regeneration was scored on the callus induction medium or after transfer of primary callus to MS + 1.0 mg L−1 BA + 0.3 mg L−1 3‐indoleacetic acid. Some individual genotypes from each of the 17 germplasm sources produced callus, and bud regeneration from callus was seen in 14 of the 17 germplasm sources, indicating that this ability is widespread in the species. Direct adventitious budding on petioles was also found within all germplasm sources. Use of BA as the sole growth regulator induced buds on callus within the range 0.1 to 3.0 mg L−1. Kinetin and tzeatin alone induced buds, but at the higher concentrations of 30 and 10 mg L−1, respectively. Environmental conditions and leaf part affected callus induction frequency on explants. Leaf blade segments were considerably more responsive than petiole segments. Explants cultured at 31°C initiated callus at least five times more frequently than those at 23°C. Highest frequency of callus induction with one genotype occurred in the dark, and no callusing occurred in 200 μmol m−2 s−1 light from fluorescent lamps. An uncomplicated procedure for shoot regeneration evolved through these experiments, permitting lone explants to initiate callus and subsequently regenerate shoots on MS + 1.0 mg L−1 BA without transfer. This system is applicable to a wtde range of B. vulgaris L. germplasm.

Greffage in vitro du pin maritime (Pinus pinaster)

Besides adventitious budding in vitro, rejuvenation of meristems could possibly be achieved by micrografting. We report here successful auto- and homo-grafting in vitro in Pinus pinaster. Apices 5–7 mm in length either (i) collected from 2- to 3-month-old seedlings or (ii) produced in vitro from an 11 -year-old tree by means of successive axillary budding cycles over a 5-year period were successfully grafted onto the epicotyl of 2- to 3-month-old seedlings. Grafted plants have been successfully transfered to field conditions. If grafted onto the hypocotyl, plants did not survive.

In vitro adventitious budding on Pinus pinaster cotyledons and needles

Adventitious budding can be induced on two types of Pinus pinaster Ait. organs. Cotyledons (10‐mm‐long), derived from 8 to 10‐day‐old seedlings, show morpho‐genetic response when an appropriate mineral solution is used (NH4+/K+= 1). Of the various cytokinin concentrations added to this optimal mineral medium, 0.8 μM BAP (6‐benzylaminopurine), with 5 nM NAA (1‐naphtaleneacetic acid), promoted organogenesis best. Buds were induced from outer mesophyll layers.Short shoots and the elongating needles (70‐mm‐long) were collected from cuttings of a mature tree (10‐years‐old). These cuttings benefited from physiological advantages of a well‐developed root system (by heating the substrate). In order to stimulate in vitro organogenesis, they had been sprayed every week from March to May with 10 μM BAP.When cultivated in the presence of 10 μM BAP and 25 nM NAA, 79% of the explants produced buds from dome‐shaped meristematic cell clusters that pre‐existed at the top of the short shoots. Moreover, among these, 42% gave rise to adventitious buds induced from proliferating mesophyll cells at the needle base. The morphologies of the two kinds of shoots were similar. Adventitious budding on these two different explants should allow vegetative multiplication of selected seedlings and elite trees.

EFFECTS OF SIMULATED ACID RAIN ON PHASEOLUS VULGARIS L. (FABACEAE)

Experiments were performed to determine the effects of simulated acid rain on Phaseolus vulgaris L. At pH values below 3, plants exhibited a failure to attain normal height, had necrotic and wrinkled leaves, excessive and adventitious budding, and premature abscission of primary leaves. Histologically, leaves had smaller cells, less intercellular space, and smaller starch granules within the chloroplasts. Respiration rates of the treated plants increased only slightly at low pH values. Apparent rates of photosynthesis, however, increased dramatically. Both carbohydrate production and root biomass were reduced by low pH treatments, and application of Congo red indicator to the acid‐treated leaf tissue showed that the cell contents were acidified to a pH of below 4.0.

Effects of Simulated Acid Rain on Phaseolus vulgaris L. (Fabaceae)

Experiments were performed to determine the effects of simulated acid rain on Phaseolus vulgaris L. At pH values below 3, plants exhibited a failure to attain normal height, had necrotic and wrinkled leaves, excessive and adventitious budding, and premature abscission of primary leaves. Histologically, leaves had smaller cells, less intercellular space, and smaller starch granules within the chloroplasts. Respiration rates of the treated plants increased only slightly at low pH values. Apparent rates of photosynthesis, however, increased dramatically. Both carbohydrate production and root biomass were reduced by low pH treatments, and application of Congo red indicator to the acid-treated leaf tissue showed that the cell contents were acidified to a pH of below 4.0.