Effects of X-irradiation on adventitious bud regeneration from in vitro leaf explants of Rosa hybrida

The effectiveness of X-radiation on regeneration of adventitious buds on in vitro leaf explants of three Rosa hybrida L. genotypes was studied. In vitro leaflet explants of roses produced adventitious buds when cultured in the dark for 1 week on Murashige and Skoog (MS) induction medium containing 6.8 μM thidiazuron (TDZ) + 0.49 μM indole-3-butyric acid (IBA) and subsequently transferred to MS regeneration medium containing 2.2 μM benzyladenine (BA) + 0.049 μM IBA in the presence of reduced light, at 15 μmol m-2 s-1 photosynthetically active radiation (PAR). Analysis of radiosensitivity by irradiating leaf explants with increasing doses of X-rays between 25 and 100 Gray (Gy) resulted in a decreasing rate of leaf explants regenerating buds from 47% to 0% respectively. The lethal dose for 50% of the regenerating explants (LD₅₀) in all the three genotypes was estimated to be 25 Gy at a dose rate 2 Gy/s. For the main experiment, doses of 5 and 15 Gy were selected and variations were observed between genotypes. Clone RUI 317 had the highest rate of adventitious bud regeneration, with 83.6% (2.5 buds/explant) at 5 Gy and 64% (1.8 buds/explant) at 15 Gy, compared to 89% (3.4 buds/explant) with the untreated control. Significant differences in the percentage of bud regeneration of the three genotypes were only observed at 15 Gy in comparison to the control and the number of buds formed per regenerating explant varied between 1 to 4. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro adventitious bud regeneration from internode segments of beech

Internode explants collected from in vitro grown shoots of two clones of Fagus sylvatica L. (European beech) and five clones of F. orientalis Lipski (Oriental beech) were used to evaluate their bud regeneration capacity. Adventitious shoot-buds formed on callus, which developed from internode segments cultured in a Woody Plant Medium supplemented with different concentrations of either thidiazuron (TDZ) or benzyladenine (BA). After 4 weeks of culture on induction media, the explants were transferred to a proliferation medium supplemented with 2.2 μM BA, 9.1 μM zeatin and 2.9 μM indole-3-acetic acid (IAA) for another 8 weeks. Medium containing TDZ was much more efficient than medium containing BA in inducing adventitious buds, the optimal TDZ concentration being 4.5 μM and the optimal BA concentration 17.8 μM. Genotypic variation in shoot regeneration capacity was observed among the two Fagus species and between clones within each species, with a significant interaction between TDZ concentration and genotype regarding mean bud number. Thidiazuron induction medium supplemented with a range of individual auxins was investigated, and it was found that IAA or indole-3-butyric acid at 2.9 μM enhanced the bud forming capacity of explants. Morphogenic response varied significantly with the position of the internode along the stem. The highest regeneration potential was obtained from apical internodes, while those distal to the apex were the least productive. Elongated shoots of adventitious origin can be readily proliferated by axillary branching. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct and Indirect Shoot Organogenic Pathways in Epicotyl Cuttings of Troyer Citrange Differ in Hormone Requirements and in their Response to Light

Bud differentiation by direct organogenesis at the apical endof Troyer citrange (Citrus sinensis[L]. OsbeckxPoncirus trifoliata[L].Raf.) epicotyl cuttings inserted vertically in a semi-solidculture medium did not require hormone additions. The numberof buds regenerated was slightly, but significantly, increasedwhen the incubation was performed in the light as compared tothe dark, and by the addition of benzyladenine (BA; 2.2 to 22µM) to the medium. Bud sprouting and subsequent shootformation required the addition of BA and was increased by lightto a higher extent than bud formation. The best response wasobtained with the highest BA concentration tested (22 µM).Regeneration through the indirect organogenic pathway at thetwo edges of the epicotyl cuttings when in contact with theculture medium did not occur in the absence of benzyladenine,which was an absolute requirement for callus development. Thebest regeneration response was obtained when the explants wereincubated in the light in the presence of 4.4 µM BA andan auxin. Indole-3-acetic acid (IAA; 5.8 µM) was moreeffective in increasing shoot formation than naphthaleneaceticacid (NAA; 0.54 µM). Higher NAA concentrations inhibitedshoot formation. Incubation in the dark or increasing the BAconcentration (22 µM) increased markedly callus growth,but inhibited both bud differentiation and sprouting, almostcompletely suppressing shoot formation. The conditions duringregeneration affected the rooting of the regenerated shoots.Rooting of 86% of the shoots was achieved in a medium with 2.7µM NAA and 2.6 µM indole-3-butyric acid. All therooted explants acclimated and survived transplanting. Underthe optimal conditions tested, the proliferation rate obtainedthrough the indirect regeneration pathway ranged from 60 to86 plants per seedling. Copyright 2000 Annals of Botany Company Troyer citrange, Citrus sinensisxPoncirus trifoliata, auxins, benzyladenine, direct organogenesis, hormone requirement, indirect organogenesis, light, morphogenesis, rooting.     

Plant regeneration from somatic tissue of Rhododendron laetum x aurigeranum

The influence of the source of plant material (greenhouse-grown plants or in vitro shoot cultures), the type of tissue explant (shoot-tip, single-node stem segment, whole leaf, leaf strip or half-leaf section) and growth regulator concentration on shoot regeneration from somatic tissue of Rhododendron laetum × aurigeranum was evaluated. No regeneration response was obtained on explants from greenhouse-grown plants. Adventitious shoots were obtained from callus produced at the basal end of shoot-tip and single-node stem segment explants derived from in vitro-grown shoots cultured on Anderson's medium supplemented with 22.8 M IAA and 73.8 M 2iP. The greatest percentage of adventitious shoot regeneration (77%) was induced on leaf sections cultured in the presence of 22.8 M IAA and 147.6 M 2iP. Plant regeneration was accomplished with minimal callus formation. This technique represents a further step toward gene manipulation of Rhododendron.Abbreviations IAA 1-H-Indole-3-acetic acid - 2iP N-(3-methyl-2-Butenyl)-1H-purin-6 amine     

Adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (<Emphasis Type="Italic">Crataegus pinnatifida</Emphasis> Bge. var. <Emphasis Type="Italic">major</Emphasis> N.E.Br.)

Hawthorn (Crataegus spp.) is an important plant with a long history as an ornamental and a source of medicine. A protocol is outlined for adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (C. pinnatifida Bge. var. major N.E.Br.). Adventitious buds were induced on both the leaves of sprouting winter buds and the leaves of in vitro plants, but the percentage of bud regeneration from leaves of in vitro plants was very low—less than 6%. On N6 medium supplemented with 31.08 μM BA and 9.67 μM NAA, the percentages of bud regeneration from leaves of sprouting winter buds of cultivars “Liaohong” and “Qiujinxing” were 31.4% and 17.6%, respectively. The regeneration abilities of three kinds of cotyledon explants, immature cotyledon, mature cotyledon, and cotyledon leaf, were compared. The percentage of bud regeneration from cotyledon leaves was higher. On MS media supplemented with 4.44 μM BA and 4.54–9.08 μM TDZ, the percentages of bud regeneration from cotyledon leaves of cultivars “Qiujinxing” and “Xiajinxing” were 27.7 ± 7.8% and 20.1 ± 4.7%, respectively, and the numbers of buds per explant were 5.9 ± 1.6 and 3.2 ± 0.7, respectively. On B5 medium supplemented with 2.22 μM BA, 2.32 μM Kn, and 0.57 μM IAA, adventitious buds grew quickly and 80–100% of buds developed into shoots. The shoots rooted successfully with the two-step rooting method. Ninety days after transplantation, more than 80% plants were survived. This system of adventitious bud regeneration from leaf and cotyledon explants could be useful for the genetic transformation and polyploidization of Chinese hawthorn.     

Plant regeneration from cultured leaves of Lavandula latifolia Medicus: Influence of growth regulators and illumination conditions

Leaves were obtained from 4-week-old seedlings of Lavandula latifolia Medicus grown in vitro. Leaf explants were then cultured on MS medium supplemented with different concentrations and combinations of the auxins IAA or NAA with the cytokinin BA and maintained under three illumination conditions, 16h photoperiod, darkness or darkness followed by a photoperiod, to assess morphogenic responses. Irrespective of illumination conditions, bud regeneration was achieved only in media containing BA or BA/auxin combinations, with the best results being obtained in the presence of BA and 0.06 or 0.6 M IAA or NAA. A photoperiod of 16h appeared to yield the best response in terms of bud regeneration percentage. High auxin concentrations (6.0 or 11.0 M) inhibited bud differentiation, especially when explants were cultured in darkness. On the other hand, low auxin levels and photoperiod improved shoot development. Excised shoots were induced to form roots by transfer to hormone-free MS medium with macronutrients at half strength. The obtained plantlets were ultimately grown in the greenhouse.Abbreviations BA benzyladenine - BM basal medium - IAA indoleacetic acid - MS Murashige & skoog - NAA -naphthaleneacetic acid     

In vitro plant regeneration from immature cotyledon explants of macadamia (Macadamia tetraphylla L. Johnson)

The macadamia tree, an Australian native, is highly valued for its nuts. Macadamia improvement programs so far have relied on conventional breeding and selection. The production of improved cultivars required to meet future demands could be accelerated by the application of modern biotechnological techniques, but this requires an efficient and reproducible regeneration system that has not yet been established for macadamia. We report here shoot regeneration from immature cotyledon explants of macadamia. Adventitious buds were induced on the cotyledon explants from fruits collected at 140 and 190 days after full bloom (DAFB) on MS medium supplemented with either 10 or 15 μM TDZ. The addition of 2% coconut milk (CM) to 10 μM TDZ containing media resulted in enhanced adventitious bud induction from 190 DAFB explants. Further shoot development from the induced buds was depressed in media containing TDZ + CM; the addition of 0.001 μM IAA to this combination doubled shoot development, from 1.9–3.9 shoots per explant. The transfer of bud clumps to media supplemented with 8.8 μM BA alone or in combination with either 0.14 μM GA₃ or 0.001 μM IAA significantly increased shoot production from the previously induced explants by 1.5–2 times of that observed in TDZ + CM medium. Histological examinations revealed that shoot regeneration was primarily by organogenesis originating from cells on or just below the cut surfaces of explants.     

Shoot regeneration from leaf explants of <Emphasis Type="Italic">Brunfelsia calycina</Emphasis>

A protocol for plantlet regeneration through shoot formation was developed for the neotropical shrub Brunfelsia calycina. This shrub is unique in its change in flower color from dark purple to white. Explants from young and mature leaves were incubated on MS medium (pH 5.7, 30 g/l sucrose, 7.5 g/l agar) with various combinations of Indole-3-acetic acid (IAA) and 6-Benzyladenine (BA) under a 16 h photoperiod at a constant temperature of 25°C. Shoot emergence was best at 4.44 μM BA and 2.85 μM IAA for young leaf explants, and at 8.88 μM BA, 2.85 μM IAA for mature leaf explants. When shoots were transferred to MS medium supplemented with 1.23–2.46 μM indole butrytic acid (IBA), they developed roots.     

Role of Light in 5-Aminolevulinic Acid Formation in Wild Strain and Mutant C-2A' Cells of Scenedesmus obliquus

In dark-grown wild strain cells of Scenedesmus obliquus, 5-aminolevulinicacid (ALA) formation was induced by irradiation with a weakblue light, as in its mutant C-2A' cells. The induction wasinhibited by distamycin A, 6-methylpurine, cycloheximide andchloramphenicol. After the light induction, the ALA formationcould proceed in the dark as well as in the light, in such heterotrophicallygrown wild type cells, but not in the greening mutant C-2A'cells. In the latter, ALA formation was dependent on red light,as well as on blue light, in the presence of CMU. The amountsof protochlorophyll in the mutant cells increased upon cessationof illumination and decreased with subsequent irradiation withblue and red light. The possible role of protochlorophyll asa photoreceptor in regulation of ALA formation in the mutantcells is discussed. ¹Present address: Laboratory of Chemistry, Faculty of Medicine,Teikyo University, Otuka, Hachioji, Tokyo 192-03, Japan. (Received January 17, 1981; Accepted April 30, 1981)     

Role of ethylene in auxin-induced flower bud formation in tobacco explants

The effect of ethytene on in vitro flower bud formation in thin-layer explants from tobacco pendicels ( Nicotiana tabacum L. cv. Samsun) was studied Endogenous ethylene production was stimulated by l-minocyclopropanc-l-carhoxylic acid (ACT), and inhibited by aminoethoxyviny lglycine (AVG). resulting in higher and lower ethylene accumulation. respectively. In the presence of an elevated ethylene concentration, the number of flower buds formed after 7 days of culture in explants was increased, compared with the control. Treatment with AVG or with AgNO₃ which blocks ethylene action resulted in decreased bud numbers after 7 days of culture. A different effect of ethylene was visible after 14 days of culture, when regeneration was complete. Treatment with AgNO₃ led to more bud regeneration, and increasing ethylene concentrations to lower bud numbers. The endogenous production of ethylene was enhanced by high concentrations of 1-naphthaleneacetic acid (NAA).
The inhibitory effect of applied ethylene was almost 100% in explants cultured at low concentrations of NAA (below 1 μ M ). but hardly visible at high concentrations (4.5 μ M ). As a consequence, the optimal NAA concentration shifted to a higher value in the presence of ethylene. These results are interpreted as a reduction in tissue sensitivity to auxin and in regenerative capability by ethylene. The effect of ethylene on auxin action is not exerted at the level of hormone concentration. Neither NAA uptake nor conversion to conjugates was effected by ethylene.     

Rapid plant regeneration from callus cultures of Plumbago zeylanica

Rapid plant regeneration was achieved in callus cultures derived from leaf and stem explants of Plumbago zeylanica Linn. on MS basal medium supplemented with 4.44 M 6-BA, 1.42 M IAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of growth regulators in the nutrient media. The leaf explants were more responsive (82.3%) than the stem explants on medium containing 1.42M IAA in combination with 4.44 M BA. The rate of regeneration was found to maintain the same level for 12 months without loss of vigour. Rooting of the differentiated shoots was achieved in media having 0.57 M IAA with 2% (w/v) sucrose within 10 days of culture. Regenerated plantlets were transferred to soil which grew normally with a survival rate of 90%. This protocol may help in the conservation of the species and selection of variants that may be induced to widen the genetic base of the genus.     

Plant regeneration from in vitro leaves of the peach rootstock ‘Nemaguard’ (<Emphasis Type="Italic">Prunus persica</Emphasis> × <Emphasis Type="Italic">P. davidiana</Emphasis>)

A complete protocol for adventitious shoot regeneration was developed from the leaves of peach rootstock ‘Nemaguard’(Prunus persica × P. davidiana) grown in vitro. Shoot explants were cultured in vitro in Murashige and Skoog medium supplemented with 3.55 μM 6-benzyladenine and 7.38 μM indole-3-butyric acid (IBA). Non-expanded leaves along with their petioles from 3-week-old in vitro-grown shoots were used as explants. Regeneration percentage was influenced by plant growth regulators, basal medium, explant type, dark period, and gelling agents. Optimal regeneration was observed with leaf explants wounded by transverse cuts twice along the midrib and first incubated with abaxial surfaces facing upward in the dark for 3 weeks, and then transferred to the light and cultured with the adaxial side in contact with regeneration medium, as seen on 1/2 MS, woody plant medium or Schenk and Hildebrandt medium supplemented with 9.08 μM thidiazuron, 0.54 μM IBA and 0.25% agar. This produced the highest regeneration percentage at 71.7% and a mean of 5.74 ± 3.24 shoots on 1/2 MS medium. Adventitious shoots were rooted (98.3–100%) and rooted plantlets survived after acclimatization to the greenhouse.     

Adventitious shoot regeneration from Begonia × erythrophylla petiole sections is developmentally sensitive to light quality

The influence of light quality on organogenesis in vitro was investigated using Begonia  ×  erythrophylla petiole explants. Pre-treatment of in vitro donor plants by growth in the dark or under far-red or blue light reduced their competence for shoot formation when compared with those grown under red or white light. Culture of competent petiole explants under far-red, blue light or in the dark reduced the number of shoots produced per explant compared to those cultured under red or white light. Explants were found to be developmentally sensitive to both far-red and blue light, because meristem, but not primordia development was inhibited. In addition, blue light inhibition of shoot formation is not mediated directly through phytochrome, as few shoots formed on explants cultured under a mixture of red and blue light which resulted in a high P _fr/ P _tot (0.82) and would allow shoot formation in the absence of blue light. Unlike the inhibitory influence of far-red light, which is reversible, exposure to blue light permanently reduces an explant's competence for shoot formation. Our results suggest that phytochrome and an independent blue light photoreceptor, possibly a cryptochrome, can regulate shoot production from B. erythrophylla petiole explants.     

Photoregulation of seed germination of wild-type and of an aurea-mutant of tomato

Seed germination of an aurea mutant of tomato ( Lycopersicon esculentum Mill.) is promoted by continuous irradiation with red, far-red or long-wavelength far-red (758 nm) light as well as by cyclic irradiations (5 min red or 5 min far-red/25 min darkness). Far-red light applied immediately after each red does not change the germination behaviour. Seed germination of the isogenic wild-type, cv. UC-105, is promoted by continuous and cyclic red light while it is inhibited by continuous and cyclic far-red light and by continious 758 nm irradiation. Far-red irradiation reverses almost completely the promoting effect of red light. The promoting effect (in the aurea mutant) and the inhibitory effect (in the wild-type) of continuous far-red light do not show photon fluence rate dependency above 20 nmol m⁻² s⁻¹. It is concluded that phytochrome controls tomato seed germination throgh low energy responses in both the wild type and the au mutant. The promoting effect of continuous and cyclic far-red light in the au mutant can be attributed to a greater sensitivity to P_fr.     

High copper levels in the medium improves shoot bud differentiation and elongation from the cultured cotyledons of <Emphasis Type="Italic">Capsicum annuum</Emphasis> L.

The effect of copper sulphate on differentiation and elongation of shoot buds from cotyledonary explants of Capsicum annuum L. cv X-235 was investigated. Shoot buds were induced on medium supplemented with 22.2 μM BAP and 14.7 μM PAA. Elongation of shoot buds was obtained on MS medium containing 13.3 μM BAP + 0.58 μM GA₃. Both shoot induction and elongation media were supplemented with different levels of CuSO₄ (0–5 μM). The levels of CuSO₄ in the induction as well as elongation medium highly influenced the shoot bud formation and their subsequent elongation. Highest number of shoot buds per explant was obtained when the concentration of CuSO₄ was increased 30 times to the normal MS level. Shoot buds formation frequency i.e., the number of shoots formed per explant was increased two fold as compared to those formed on control. Elongation both in terms of percentage and length of shoots was better than that on control. Healthy elongated shoots were rooted on MS medium supplemented with 5.7 μM IAA. Rooted plantlets were transferred to field conditions.     

Rapid in vitro multiplication of <Emphasis Type="Italic">Melia azedarach</Emphasis> L. (a multipurpose woody tree)

An efficient regeneration protocol for rapid multiplication of Melia azedarach, an economically as well as medicinally important timber-yielding tree, was developed. Nearly 90% of the culture exhibited axillary bud sprouting and multiple shoot formation from nodal segments derived from 20-year-old candidate plus tree on Murashige and Skoog (MS) medium supplemented with 5 μM 6-benzyladenine (BA). The highest shoot regeneration frequency (92%), maximum number of multiple shoots (19.7 ± 0.31) as well as shoot length (4.9 ± 0.08 cm) was induced from nodal explants on MS medium amended with 5.0 μM BA, 0.5 μM indole-3-acetic acid (IAA) and 30 μM adenine sulfate (AdS). Addition of 250 mg l⁻¹ ammonium sulphate, (NH₄)₂SO₄, and 100 mg l⁻¹ K₂SO₄, prevented defoliation and tip burning without affecting the number of shoots. The explant harvest period also influenced the bud break and shoot sprouting from nodal segments. Repeated subculturing of nodal explants on fresh MS medium containing lower concentration of BA (2.5 μM) along with IAA (0.5 μM), AdS (30 μM) and additives was found most suitable growth regulator regime for achieving 1.2-fold increase in shoot multiplication rate. The percentage of shoot multiplication as well as the number of shoots per node remained the same during first three subculture passages, afterwards a decline was recorded. About 90% of the in vitro regenerated shoots were successfully rooted ex vitro by giving a pulse treatment of 250 μM indole-3-butyric acid for 15 min, followed by their transfer to thermocol cups containing soilrite. The raised plantlets were successfully acclimatized first under culture room conditions, then to green house with 85% survival rate.     

Interactive effect of light,temperature and TDZ on the regeneration potential of leaf discs of <Emphasis Type="Italic">Fragaria x ananassa</Emphasis> Duch

This is the first report where shoot regeneration in strawberry cultivar Chandler has been achieved simultaneously through both somatic embryogenesis and shoot bud formation. Direct somatic embryogenesis was observed in leaf discs which were cultured on medium containing MS salts + B₅ vitamins + 2% glucose + 18.16 μM thidiazuron (TDZ) and given both chilling and dark treatment for 2 wk at 4 ± 2°C followed by incubation at 25 ± 2°C under 16-h photoperiod for third wk. After 3 wk, these explants were then subcultured on medium containing MS salts + B₅ vitamins + 2% glucose and incubated under 16-h photoperiod at 25 ± 2°C for further growth and development. Direct regeneration via de novo shoot bud formation was observed in leaf disks which were given dark treatment and were cultured on medium containing MS salts + B₅ vitamins + 2% glucose supplemented with 9.08 μM TDZ. There was a synergistic effect of photoperiod, dark, and chilling treatments on somatic embryogenesis, whereas chilling treatment had an inhibitory effect on shoot organogenesis.