Lepoivre Medium Research Articles

Root Regeneration on a Protoclone Issued from Root Callus of P.cerasus cv. Montmorency

ABSTRACTA fast growing callus obtained after cultivating root sections of 1 sm lenght on Murashige and Skoog (MS) medium with 2 mg/l 1- naphtalene acetic acid (NAA) and 0.25 mg/l 6-benzyl- aminopurine (BAP) (pH 5.6) as the hormonal balance, was used as the source of protoplasts. Suspensions of 107 protoplasts/g of fresh weight with 94% viability were cultivated on the basic MS medium supplemented with 9% (W/v) mannitol and 2 mg/l NAA, 0.5 mg/l BAP, 0.5 mg/l zeatin (pH 5.6) for further cell wall regeneration, cell colonies formation and microcalli growth. One root regenerated after subcultivating the calli in MS medium with various combinations of NAA, BAP and Zeatin. A second root regenerated after further calli subcultures on Lepoivre medium only containing 2 mg/l indole-3-butyric acid. The paper dwells on the determinant factors for the process of rhizogenesis as an intermediate step towrds obtaining shoot buds on a cherry-tree.

Regeneration of dihaploid chicory (Cichorium intybus L. var. foliosum Hegi) via microspore culture

AbstractIn order to develop fully inbred chicory plants, dihaploid plants were raised from callus derived from microspores of three selected Witloof, Robin and Treviso types. Microspores were isolated from florets containing pollen at the uninuclear state and cultured in a modified MS medium plus 0.5mg/l 2,4‐D, 0.5mg/l IAA and 2.0mg/l zeatin. During culture periods of up to 6 months, gametoplasts emerged from pollen grains, divided and started to form colonies and calli. These were subcultured on the same basal medium supplemented with 0.5mg/l BA and 0.5mg/l IAA. Shoot growth was enhanced on a low salt‐containing medium supplemented with 0.4mg/l kinetin and 0.2mg/l IAA. Shoots were rooted on a half‐strength Lepoivre medium plus 0.2mg/l IBA and finally transferred to soil. Florets were excised from 34 capitula, but only microspores from four of them developed into plants via callus. More than 450 plants were raised in the greenhouse and the field. Leaves from these plants were subjected to DNA fluorescence analysis via flow cytometry: a range of ploidy levels was detected. The cell composition of 44 of these plants was predominantly haploid, with a diploid background. Regenerant plant phenotypes were compared with the parent genotypes. The value of such haploids in commercial chicory breeding is discussed.

Organogenesis and somatic embryogenesis in Cupressus sempervirens

Adventitious buds of Cupressus sempervirens L., were formed on excised mature embryos cultured for 10 days on half-strength Quoirin and Lepoivre medium (1/2QP) with 10 μM N6-benzyladenine. For shoot development, embryos were transferred to 1/2QP without growth regulators. Axillary shoot formation and rooting occurred spontaneously as adventitious shoots aged and transfer intervals were increased. Embryogenic tissue was obtained from immature embryos on induction media consisting of von Arnold and Eriksson (AE) or Gupta and Durzan (DCR) salts with 10 or 20 μM 2,4-dichlorophenoxyacetic acid. Cultures were maintained on DCR with 5 μM α-naphthaleneacetic acid and 5 μM BA.

576 PB 053 METHODS TO MAXIMIZE AND MINIMIZE SEGREGATION OF CHIMERAL PEAR (PYRUS SPP) IN VITRO

Variegated `Louise Bonne' (LB) pear is a periclinal chimera in which the LIII layer is albino. Chimeral shoots propagated in vitro segregate spontaneously into green, albino, pale, or rearranged chimeral types, making them difficult to maintain in culture. We investigated the role of growth regulators on chimeral stability and destability to find a combination that would maintain the chimera through repeated subcultures. 70 to 90% of shoots remained chimeral on Lepoivre (LP) medium supplemented with 8 μM BA or less. Only 36 to 58% of shoots grown at concentrations greater than 8 μM were stable. Shoots grown on LP with thidiazuron (TDZ) were very unstable (4 to 44%). NAA had no significant effect on chimeral stability. While shoots multiplied better on LP, the chimeral pattern was more obvious on MS, making it a good screening medium. Selection and subculturing chimeral shoots on a good medium (LP with 2 to 4 μM BA) increased the percentage of chimeral shoots from 26% at the 4th subculture to 84% at the 27th subculture.

Period of harvest, sprouting ability of cuttings, and in vitro plant regeneration in Fraxinus excelsior

Propagation by stem cuttings and in vitro culture of apical bud explants were studied on Fraxinus excelsior L. Stem cuttings from 4- to 7-year-old trees growing under natural conditions sprouted only when cuttings were taken from dormant material. Only 6% of those that had sprouted developed roots by the 7th month of culture. Similarly, only apical bud explants harvested during the dormant period sprouted in vitro. Up to 87% of these sprouts developed two to four branching adventitious roots after 5 months of culture. During the initial phase of in vitro culture, the Quoirin and Lepoivre medium and the woody plant medium favoured sprout lengthening. During the phase of multiplication, up to three sprouts per explant developed with the woody plant medium in the presence of a combination of high 6-benzylaminopurine (3.0–4.0 mg∙L−1) and low indole-3-butyric acid (0.01–0.03 mg∙L−1) concentrations. Rooting was obtained in a medium without any growth regulators. Microscopic analysis showed a direct connection between the vascular elements of adventitious roots and stem of plantlet. Chromosome number in root apices of ash plantlets and ash trees grown under natural conditions was 2n = 46. Key words: chromosome number, Fraxinus excelsior L., in vitro plants, micropropagation, stem cuttings.

IN VITRO MAINTENANCE OF CHIMERAL PEAR

Variegated `Louise Bonne' (LB) pear shoots propagated in vitro tend to segregate spontaneously into green, albino, pale, or rearranged chimeral types. This instability makes it difficult to maintain LB in its original chimeral form. This study was initiated to investigate the role of growth regulator combinations on chimeral stability and to find a combination that could be used to maintain the chimera through repeated subcultures. Shoots were most stable (70 to 90% remained chimeral) on LePoivre (LP) medium supplemented with less than 8 μM BA. Shoots grown at concentrations greater than 8 μM were less stable (37 to 58%). Cultures grown with thidiazuron (TDZ) were very unstable (7 to 46%). NAA at the highest concentrations (5 and 10 μM) decreased chimeral stablity both alone (40 and 8%, respectively) and in combination with TDZ (7 to 16%). The implications of growth regulator combinations on long-term chimeral maintenance in vitro will be discussed.

In vitro shoot multiplication of eastern white cedar (Thuja occidentalis)

A protocol for clonal propagation of eastern white cedar (Thuja occidentalis L.) was enhanced by optimizing the shoot multiplication stage using unbranched in vitro-produced shoots. This was achieved by careful selection of different medium components. An optimum range of 10 to 14 axillary shoots was obtained when shoots were cultured on half-strength Quiorin and LePoivre medium containing 10µM filter-sterilized zeatin for 3 wk. Transfer of the treated shoots to cytokinin-free medium containing 0.05% activated charcoal improved both the number and quality of the axillary shoots produced. Maximum axillary bud induction was also accomplished when shoots were pulsed in 1 mM liquid, filter-sterilized zeatin for 3 h, and then transferred to half-strength Quiorin and LePoivre, charcoal-containing medium. Inclusion of 4% sucrose improved the number of axillary shoots obtained. Half strength of the major salts produced an optimum response. Shoots obtained from different cultures (1 to 5 yr old) responded similarly to the applied cytokinin; however, newly induced shoots (4 mo. old) gave a significantly higher response.

Influence of taxonomic relatedness and medium composition on meristematic nodule and adventitious shoot formation in nine pine species

Embryos of Pinusechinata Mill., Pinustaeda L., Pinusserotina Michx., Pinuseldarica Medwed., Pinuscaribaea Morelet, Pinusoocarpa Scheide, Pinustecunumanii (Schwd.) Equiluz & Perry, Pinusstrobus L., and Pinusradiata D. Don were cultured following the protocol for Pinusradiata to determine if a perpetual meristem culture could be produced. Two subsequent experiments were done that included modifications to the medium's mineral composition, strength (modified LePoivre and Murashige and Skoog at half-strength and full strength), cytokinin concentration (1.0, 2.5, and 5.0 mg/L benzyladenine), and auxin concentration (0 and 0.1 mg/L indolebutyric acid). Pines in subsection Australes performed poorly in culture relative to those species in subsection Oocarpae. Pinusradiata and Pinuseldarica were the only species to produce long-term subculturable meristematic tissue, although shoots were obtained with seven of the species. Half-strength modified LePoivre medium containing 2.5 mg/L benzyladenine but no auxin gave the best results with most species. Significant differences in shoot production were found among Pinusoocarpa provenances.

Plantlet multiplication from white pine (Pinus strobus L.) embryos in vitro: bud induction and rooting

White pine embryos were grown on 4 different media with 6 different benzyladenine (BA) concentrations. Maximum adventitious shoot initiation and growth were obtained on a modified Lepoivre medium with 20 μM BA. Modified Schenk and Hildebrandt, Murashige and Skoog, and Gresshoff and Doy media were also tested. Shoot elongation was achieved on half-strength basal medium lacking growth regulators. Three rooting experiments involving indole-3-butyric acid (IBA), sucrose concentration, shoot orientation, IBA pulse length, and light or dark were carried out. Treatment of shoots in an upright position with 50 μM IBA for eight days followed by culture in a medium with 3% sucrose in the light produced the most rooting (50% at 3 months). Rooted shoots were transplanted to the greenhouse for further growth.

Adventitious shoot regeneration from leaf tissue of three pear (Pyrus sp.) cultivars in vitro

To develop an adventitious regeneration system for pear cultivars, several experiments were conducted with 2 cultivars of Pyrus communis L. ('Seckel' and 'Louise Bonne') and one cultivar of P. bretschneideri Rehd. ('Crystal Pear'). Half-leaves, taken from shoots proliferating on Lepoivre medium, were plated in petri-dishes on medium supplemented with various combinations of cytokinins and auxins. Cultures of the above cultivars had been established from mature trees. Among the growth regulators tested, thidiazuron (TDZ), combined with naphthalene-acetic acid (NAA), was the most efficient for stimulation of adventitious shoots. The optimum level of TDZ was about 3 uM; shoot regeneration was observed over a wide range of TDZ and NAA concentrations (0.5 to 5 uM and 2.5 to 13 um, respectively). Among different macronutrient compositions, 1/2 and 1/4 Murashige and Skoog were the most effective. Sucrose concentrations (10 to 50 g L-1) had a linear significant effect on shoot regeneration of 'Crystal Pear'.

Relationship between Indole-3-Acetic Acid Levels in Apple (Malus pumila Mill) Rootstocks Cultured in Vitro and Adventitious Root Formation in the Presence of Indole-3-Butyric Acid

In vitro rooting response and indole-3-acetic acid (IAA) levels were examined in two genetically related dwarfing apple (Malus pumila Mill) rootstocks. M.26 and M.9 were cultured in vitro using Linsmaier-Skoog medium supplemented with benzyladenine (BA), indole-3-butyric acid (IBA), and 1,3,5-trihydroxybenzoic acid (PG). Rooting response was tested in Lepoivre medium supplemented with IBA and PG. IBA concentrations of 12.0 and 4.0 micromolar induced the maximum rooting percentages for M.9 and M.26, respectively. At these concentrations rooting response was 100% for M.26 and 80% for M.9. Free and conjugated IAA levels were determined in M.26 and M.9 shoots prior to root inducing treatment by high performance liquid chromatography with fluorescence detection and validated by gas chromatography-mass spectrometry using (13)[C(6)]IAA as internal standard. Basal sections of M.26 shoots contained 2.8 times more free IAA than similar tissue in M.9 (477.1 +/- 6.5 versus 166.6 +/- 6.7 nanograms per gram fresh weight), while free IAA levels in apical sections of M.26 and M.9 shoots were comparable (298.0 +/- 4.4 versus 263.7 +/- 9.3 nanograms per gram fresh weight). Conjugated IAA levels were significantly higher in M.9 than in M.26 indicating that a greater proportion of total IAA was present as a conjugate in M.9. These data suggest that differences between M.26 and M.9 rooting responses may be related to differences in free IAA levels in the shoot base.

In Vitro Germination and Growth of Rubus Seeds and Embryos

Abstract Freshly harvested seeds of 10 Rubus crosses were germinated in vitro on modified Lepoivre medium without growth regulators. Among several seed preparation treatments, the best germination and subsequent survival in soil was achieved with halved seeds. In vitro germination bypassed the need for cool moist stratification and resulted in 57% to 81% germination within 8 to 12 days. This system provides an alternative method to secure high germination when a breeder is working with limited seed number, as in some interspecific Rubus crosses.