Adventitious Regeneration Research Articles

The Biomass Dosage Influences the Effects of Diethyl Aminoethyl Hexanoate on Micropropagation of Echinacea purpurea (L.) Moench

The plant growth regulator diethyl aminoethyl hexanoate (DA-6) has proved highly effective on micropropagation of the medicinal plant purple coneflower (Echinacea purpurea (L.) Moench), however, sharp variation of the effects existed among explants in the same treatment, making the application of DA-6 in micropropagation difficult. In order to clarify factors that influencing the treating results of DA-6, explants with different biomass dosage were prepared and inoculated onto medium supplemented with different concentrations of DA-6. It was found that among the three kinds of biomass dosage explants, the lowest biomass explants required the lowest concentration of DA-6, and the highest biomass explants required the highest concentration of DA-6 for the best results on adventitious buds regeneration. Similar results were obtained when regenerated buds of three different biomass dosages were cultured. It could be concluded from the above experimental results that for achieving better DA-6 application results, the concentration of DA-6 should be determined not only by the types but also by the biomass dosage of the explants. The present finding might help to improve the micropropagation efficiency in E. purpurea, and might be applicable for other species

Tryptophan metabolism and evaluation of morphological, biochemical and molecular variations in a field grown plant population derived via direct adventitious shoot bud regeneration from pre-plasmolysed leaves of Catharanthus roseus

Sixty six plants raised via direct shoot bud organogenesis from pre-plasmolysed leaf explants of Catharanthus roseus were assessed under in vivo conditions for their physio-morpho traits, tryptophan metabolism, genetic fidelity and alkaloid profile. Morphologically all plants were parent like except the three morpho-types i.e. broad cup-shaped leaves, dwarf phenotype and enlarged pink corona. Vindoline was detected in most of the plants in substantial amount while ajmalicine, ajmaline and catharanthine were detected in few plants only. More than eightfold increased vindoline (0.08 % dry wt.) was showed by plant no. 1, 4 and 39. Except plant no. 54, which accumulated the highest alkaloid content of 3.09 % dry wt. with tryptophan content of 0.0283 % dry wt., majority of the plants showed a negative correlation between total alkaloid content and tryptophan accumulation and strong positive correlation between tryptophan content and 5-methyltryptophan tolerance. ISSR and RAPD profile of seventeen randomly selected, field established plants was generated by using 10 ISSR and 60 RAPD primers. In RAPD, a total of 753 bands were detected, out of which 624 (82.87 %) were monomorphic. In ISSR profiling, a total of 205 bands were detected out of which 200 bands were monomorphic (97.56 %) and only 5 bands were found to be polymorphic. Highly significant data for the monomorphic banding pattern across all the three genotypes (p < 0.01) was observed. Principal components, AMOVA analysis and multivariate analysis using Nei and Li’s coefficient further validate the monomorphism. Thirteen plants having superior traits were grown in Random Block Design in field. The relevance of physiological and epigenetic changes occurred in the light of morpho-types and alkaloid profile of directly regenerated plants during in vitro to in vivo acclimatization in C. roseus is discussed.

IN VITRO REGENERATION OF ANNATTO (BIXA ORELLANA L.) PLANTLETS FROM NODAL AND INTERNODAL ADULT STEM SEGMENTS

This work aimed to evaluate the in vitro morphogenic response of adult explants of Bixa orellana and to establish a regeneration protocol from adult nodal and internodal explants from 16-year-old adult plants propagated by air-layering. For internodal segments the following treatments were used: T1, MS medium lacking growth regulators (MS0); T2, MS + 4.92 µM 6-I³,I³-dimetylalylamino-purine (2-iP); T3, MS + 4.56 µM Zeatin (ZEA); T4, MS + 4.44 µM 6-benzyladenine (BA); T5, MS + 4.54 µM Thidiazuron (TDZ), with ten repetitions for each treatment. Thirty days after establishment of INS, better regeneration rates were achieved with ZEA and TDZ, when compared to BA, 2-iP and MS0. For nodal segments the best results were obtained using the combination of 2iP and BA. In conclusion, the promotive effect of direct adventitious organogenesis from adult internodal segments or axillary shoot proliferation from adult nodal segments, in B. Orellana, is dependent on the type and concentration of cytokinin used. Histological analyses showed the presence of meristemoids in the regenerated tissue. This region was characterized by the typical organization of the vascular system into a cylindrical structure, with central xylem, and with fast proliferating and radially distributed cells. Our analyses also revealed that all budding activity had a vascular system connected to the original explant, confirming the organogenic pathway-related ontogeny. We suggest the use of MS medium supplemented with cytokinins ZEA or TDZ and 2iP or BA for regeneration of annatto from INS and NS, respectively, which will reduce the regeneration time of this species in vitro. The protocol could be further used in transformation systems applied to adult elite genotypes to improve bixin production. This is the first report on adventitious regeneration from adult explants of annatto.

Efficient culture protocol for plant regeneration from petiole explants of physiologically mature trees of Jatropha curcas L.

An efficient and reproducible protocol for induction of adventitious shoot buds and plant regeneration from petiole explant cultures of Jatropha curcas, an important biofuel crop, is described. Physiologically mature trees of three J. curcas genotypes were selected and explants were prepared from young petioles. Treating the explants with high concentrations (5 to 120 mg/L) of thidiazuron (TDZ) solution for short time periods (5 to 80 min) helped increase the regeneration frequency and improved the quality of the regenerated buds significantly. The age of the petioles and inoculation methods were found to influence the culture results. The best shoot buds induction (65.78%) and number of buds (6.77) per explant was seen in the second petiole explants of genotype M-1 treated with 20 mg/L TDZ solution for 20 min, followed by 35-day culture on hormone-free Murashige and Skoog medium. The regenerated buds could elongate to become shoots in a medium containing gibberellic acid. The elongated shoots initiated roots to become intact plantlets in rooting medium containing indole-3-butyric acid and L-glutamine (Gln), and supplementing 16 mg/L Gln into the rooting medium effectively stimulated the initiation and growth of roots, with the best rooting rate (51.72%). After acclimatization, these plantlets were transplanted to soil wherein normal growth was observed. Therefore, an intact plantlet could usually be obtained at 60 days of culture by using the culture protocol described in this study. This protocol can be used for mass production of true-to-type plants and the production of transgenic plants through Agrobacterium/biolistic-mediated transformation.

A calamitalean forest preserved in growth position in the Pennsylvanian coal measures of South Wales: Implications for palaeoecology, ontogeny and taphonomy

A newly discovered fossil forest is reported from Lower Pennsylvanian (late Langsettian) strata in the classic Amroth to Wiseman’s Bridge section of Pembrokeshire, South Wales. It comprises nearly two hundred sediment-cast Calamites axes and putative groundcover ferns, preserved in growth position in the deltaic mouth bar deposits of a marine-influenced coastal plain. Vegetation-Induced Sedimentary Structures developed around upright axes imply that dense Calamites thickets promoted localized patterns of sedimentation, and ultimately, stabilized substrates, paving the way for the establishment of mire vegetation. Not long after initial colonization, a short-lived brackish incursion resulted in dieback of the Calamites thickets; however, a few axes survived to recolonise the delta front through adventitious regeneration, implying that calamitaleans were, to a limited degree, saline tolerant. A remarkable feature of the fossils is that many examples preserve an internal pith-cast embedded within a stem-cast. Preservation of both pith- and stem-casts for the same plant allows inference of an ontogenetic growth-series, and shows that these clastic-substrate Calamites were much more substantially ‘woody’ than previously conjectured. This raises the possibility that plastic developmental traits like growth rate and longevity may have been what mainly distinguished these opportunistic Calamites in disturbed clastic settings from their much larger, and even more woody, cousins in adjacent mires. Analysis of the fossils also contributes to taphonomic debates, supporting the hypothesis that upright Calamites axes are mostly preserved as stem-casts (rather than pith-casts, as traditionally believed). As such, the newly discovered fossil forest sheds light on the ecology, ontogeny, and taphonomy of Calamites – an iconic, familiar, yet, insufficiently understood Pennsylvanian plant.

Adventitious regeneration in styrian oil pumpkin in relation to the endoreduplication pattern and induced tetraploidy on fusaric acid-supplemented media

We attempted to improve the adventitious regeneration protocol in styrian oil pumpkin for subsequent use in somaclonal variation studies. The endoreduplication status of tissues might play an important role, so endopolyploidy was analyzed by flow cytometry in different organs and major differences were revealed. Endoreduplication was minimal in leaves but in other organs—particularly in the hypocotyl and epicotyl—levels up to 64C were found. The basal part of cotyledons has been reported to be the most responsive for adventitious regeneration and, using image cytometry, we identified it as the least endoreduplicated section, with a cycle value of 1.29 compared to 1.78 and 1.80 in the central and distal parts. Basal cotyledonary explants were subjected to various media combinations, revealing Murashige and Skoog (Physiol Plant 15:473–497, 1962, doi:10.1111/j.1399-3054.1962.tb08052.x) medium supplemented with 1 mg L−1N6-benzylaminopurine (BA), 0.25 mg L−1p-aminobenzoic acid (PABA) and 5 mg L−1 fusaric acid (FA) to be optimal (73.3 % regenerated explants with 1.8 shoots per regenerating explants). FA was added to media as a possible selective agent for somaclonal variation inducing increased tolerance to Fusarium but was surprisingly found to stimulate regeneration at a low concentration (5 mg L−1) and to induce genome doubling at medium concentrations (10 and 20 mg L−1).

Effect of meta-Topolin on micropropagation and adventitious shoot regeneration in Prunus rootstocks

The effect of meta-Topolin (mT), an aromatic natural cytokinin, on micropropagation and adventitious shoot regeneration was evaluated on Prunus rootstocks, Torinel (Prunus domestica L.) and Ferdor (Prunus insititia × domestica). In vitro shoots were grown for three subcultures on a multiplication medium containing 2.1, 4.2 or 6.3 μM of mT or 2.1 µM N 6-benzyladenine (BA). Then, apical leaves were excised and transferred on a medium supplied with BA, thidiazuron (TDZ) or zeatin for adventitious regeneration. Shoots multiplied on 2.1 μM mT or BA, were also induced to root with α-naphthalene acetic acid and acclimatized. meta-Topolin did not improve shoot proliferation, respect to BA, however, positively influences growth and quality of shoots. Ferdor from mT showed higher rooting percentage (92 %), root number and length, respect to the control, while a similar response was observed in Torinel with both cytokinins. Acclimatisation was higher than 90 % for both genotypes and, after 5 months, the highest length of roots was found in plants from mT. Adventitious regeneration was obtained only in leaves from shoots previously grown on mT. The highest regeneration responses, 65 and 42 %, respectively for Ferdor and Torinel, were obtained in the regeneration medium supplied with TDZ.

Adventitious Shoot Organogenesis and Plant Regeneration from Leaf and Cotyledon Explants of Citrullus colocynthis

Plant regeneration protocol for Citrullus colocynthis L. was achieved from leaf and cotyledon explants. Explants were cultured on MS medium supplemented with 2,4-D, in combination with BAP,TDZ, Kn and Zeatin. Leaf explants are the best source for the induction of high percentage of caulogenesis and regeneration (45%) on MS + 2.0 mg.L−1 Kn + 1.0 mg.L−1 TDZ. In cotyledon derived callus cultures 2,4-D + Zeatin combination played an important role in induction of multiple shoots (15–18) with 50% of regeneration response. Each cotyledon explants produce an average number of shoots (9.75 ± 0.2) with average shoot length 3.66 ± 0.1.

Adventitious bud regeneration from cotyledonary node, hypocotyl, cotyledon and euphylla of various genotypes of sunflower (Helianthus annuusL.)

Isolated culture of sunflower is significantly important in seeking new species of sunflowers with plenty of essential oil by modern biotechnologies. Thus, the callus development and adventitious bud regeneration from various explants of sunflower were studied by in vitro tissue cultures. It was found that the callus were readily formed on six genotype explants cultured in media with IAA and 6-BA, but adventitious bud regeneration from these callus was relatively weak. The adventitious bud regeneration inductivities of four tissue explants from different genotype sunflowers were ordered as followed: cotyledonary nodes > hypocotyls > cotyledons > euphyllas. The desired concentrations of 6-BA inducing adventitious bud regeneration were at 1.2–1.8 mg/l for cotyledonary nodes, 1.8 mg/l for hypocotyls and 0.6 mg/l for cotyledons, respectively. But no adventitious buds were differentiated from euphyllas only by 6-BA. The best medium for organogenesis was composed of MS+0.03 mg/l IAA+1.2–1.5 mg/6-BA by which euphyllas also were induced to regenerate adventitious buds with an averagely 20.83% inductivity. Among several genotype sunflowers, PR29 had the highest regeneration frequency and the regenerated plantlet has been successfully cultured.

Development of a seedling clone with high regeneration capacity and susceptibility to Agrobacterium in apple

Agrobacterium-mediated transformation has been the preferred system for transforming apples. Although transgenic lines have been obtained form a number of genotypes, the transformation efficiency was not high. Apple is highly heterozygous genetically, so its seedling populations segregate heavily. We speculate that a genotype with high regeneration capacity and sensitive to Agrobacterium could be screened from the seedling population of apple. In order to verify this hypothesis, we developed in vitro seedling clones of apple (Malus×domestica) and compared their regeneration capacity and susceptibility to Agrobacterium tumefaciens in this study. Royal Gala, a genotype had been widely used in apple transformation, was selected as the source of seeds. First, 10 clones with higher multiplication capacity and normal leaf shape and smooth leaf surface, named GL-1 to GL-10, were screened out from 100 in vitro seedling clones. The capacity of adventitious bud regeneration from leaf segments were compared among the ten clones, and the result showed that GL-3 exhibited extremely higher adventitious regeneration capacity. GL-3 was also compared with its parent on the regeneration capacity, and the data showed that the number of buds per explant of GL-3 was 2.6 fold higher than that of Royal Gala. The susceptibility of GL-3 to Agrobacterium was compared with Royal Gala by monitoring the transient expression of GUS in leaf segments. For GL-3, the percentage of explants with large GUS-positive patches was 46.7±1.1%, which was obviously higher than that (25.1±7.2%) of Royal Gala. After the leaf segments of GL-3 were transformed with A. tumefaciens strain EHA105 carrying the AtmiR156a antisense gene, the percentage of explants with kanamycin-resistant buds reached 27.5%. In conclusion, a genotype with high regeneration capacity and sensitive to Agrobacterium was screened out from the seedling population of apple, and this strategy could be applied in other perennial woody plants.

Plant regeneration from immature seeds of Eugenia myrtifolia Sims.

Eugenia myrtifolia Sims. is an evergreen shrub, native to temperate and tropical rainforests of Australia, which is becoming an important containerized ornamental plant in the US and Mediterranean nursery industries. To satisfy the growing market demands for this new ornamental plant, development of an accelerated propagation method is required. The goal of this study was to investigate the in vitro regeneration potential of E. myrtifolia Sims. seeds at different stages of development, towards establishment of an in vitro multiplication system. Maximum regeneration of adventitious shoots was achieved from immature seeds cultured in the dark on half-strength Murashige and Skoog (MS) macronutrients and full-strength MS micronutrients and vitamins (MS/2) medium supplemented with 2.5 μM thidiazuron (TDZ). Induction of regeneration occurred after at least two successive subcultures on TDZ-enriched medium, followed by subcultures on expression medium (hormone free MS/2) or multiplication medium [MM; MS medium enriched with 4.4 M 6-benzyladenine and 0.05 M α-naphthaleneacetic acid], where complete development of shoots occurred. The regenerated shoots were excised and transferred again onto MM for micropropagation, where a proliferation rate of 1:4 was achieved, and finally the shoots were transferred to a hormone-free MS medium for rooting. Following ex vitro transplanting, acclimatization over a period of 15 d was sufficient to establish greenhouse plants. The regenerated plants grown in the field for more than 2 yr showed the same phenotype as that of mother plants. The adventitious regeneration and micropropagation carried out in this study can be used for a large-scale propagation and genetic engineering of E. myrtifolia Sims.

Phosphomannose-isomerase as a selectable marker for transgenic plum (Prunus domestica L.)

A mannose selection system was adapted for Agrobacterium-mediated transformation of plum (Prunus domestica L.) hypocotyl explants and the recovery of transgenic plants. Adventitious regeneration from non-transformed hypocotyl sections was inhibited when 3 mg/l mannose, combined with 10 mg/l sucrose, was added to the medium. Mature seed hypocotyl slices from the cultivar ‘Claudia Verde’ were infected with A. tumefaciens AGL1, carrying the pNOVgus vector, and placed onto different selective media with mannose. A low mannose selection (1.5 g/l, regeneration below the inhibitory concentration) applied for 16 weeks led to the regeneration of escapes. However, when mannose at 1.5 g/l or at 3 g/l (the regeneration-inhibiting concentration) was applied for 6 weeks from the beginning of the experiments and, after that, was increased to 5 g/l, several independent transgenic lines were obtained. The transformation events were monitored by detection of the GUS enzymatic activity at different stages of the process. Nevertheless, stable integration of transgenes into the genome of the plum plants was confirmed by PCR and Southern blot analysis. The transformed shoots were rooted on a medium supplemented with 10 g/l sucrose and 4 g/l mannose. The transformation procedure described here, using the pmi/mannose system for selection of transgenic plum plants, represents an alternative for the production of transgenic plum plants under conditions that are safe regarding human health and the environment, and would permit the insertion of more transgene/s in a pre-existing transgenic line.

Acceleration of Adventitious Shoots by Interaction Between Exogenous Hormone and Adenine Sulphate in Althaea Officinalis L.

In the current study attempts were made to investigate the effects of three different phases of callus induction followed by adventitious regeneration from leaf segments (central and lateral vein). Callus induction was observed in Murashige and Skoog's (MS) medium supplemented with 15.0μM 2,4-dichloro phenoxy acetic acid (2,4-D). Adventitious shoot buds formation was achieved on MS medium supplemented with 7.5μM 2,4-D and 20.0μM AdS in liquid medium as it induced 19.2 ± 0.58 buds in central vein explants. Addition of different growth regulators (cytokinins-6-benzyladenine, kinetin and 2-isopentenyl adenine alone or in combination with auxins-indole-3-acetic acid, indole-3-butyric acid and α-naphthalene acetic acid, improved the shoot regeneration efficiency, in which 5.0μM 6-benzyl adenine along with 0.25μM α-naphthalene acetic acid was shown to be the most effective medium for maximum shoot regeneration (81.3%) with 24.6 number of shoots and 4.4 ± 0.08cm shoot length per explant. Leaf culture of central veins led to better shoot formation capacity in comparison to lateral vein. Rooting was readily achieved on the differentiated shoots on 1/2 MS medium augmented with 20.0μM indole-3-butyric acid. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 80% survival rate.

Anatomical observations of adventitious bud regeneration from leaf explants of Ziziphus jujube Mill. ‘Huizao’

The histological process of adventitious shoot regeneration from the leaf explants of Zizyphus jujuba ‘Huizao’ was reported in this study. This is the first report on the detailed histological process of direct shoot regeneration from leaf explants in Z. jujube. Shoot regeneration was obtained from woody plant medium (WPM) supplemented with 2.27 μM thidiazuron (TDZ), 1.07 μM α-naphthalene-acetic acid (NAA) and 2.94 M silver nitrate (AgNO3) for 10 days in the dark followed by 3 weeks at a 14 hours photoperiod. The adventitious buds mostly formed from leaf veins and petioles, and the further histological studies revealed that there were multiple vascular bundles around leaf veins and the adaxial side of explants, and the adventitious buds directly originated from the parenchymatous cells around the vascular bundles without the intervening callus phase. After 3 days of culture, the parenchymatous cells started dividing and meristemoids formed thereafter. The meristematic cells continued division and subsequently gave rise to bud primordia. Well-developed shoot buds through direct organogenesis was achieved after 20 days of culture.

Ploidy level stability of adventitious shoots of sour cherry ‘Čačanski Rubin’ and Gisela 5 cherry rootstock

The capacity of regeneration of adventitious shoots from leaf explants was studied in sour cherry ‘Cacanski Rubin’ (Prunus cerasus L.) and cherry rootstock Gisela 5 (P. cerasus × P. canescens). Regeneration assay included thirty different combinations of plant growth regulators. 6-benzyladenine (BA) and thidiazuron (TDZ) were applied either individually or each combined with different concentrations of indole-3-butyric acid, α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). ‘Cacanski Rubin’ showed higher regeneration capacity in comparison with Gisela 5 regarding the total number of treatments inducing regeneration as well as the highest frequency of regeneration achieved. In both genotypes, 8.9 μM BA was more effective than both 4.5 and 9.0 μM TDZ in inducing adventitious regeneration, but only when combined with auxins. The highest frequency of regeneration (20.8 %) in ‘Cacanski Rubin’ was achieved on medium supplemented with 8.9 μM BA combined with 5.4 μM NAA, while in Gisela 5 the highest value (8.3 %) was obtained when BA was combined with 4.5 μM 2,4-D. Flow cytometry combined with 4′-6-diamidino-2-phenylindole staining was employed to estimate DNA ploidy levels and relative nuclear DNA content in adventitious regeneration-derived shoots, in vitro shoots of axillary origin and in vivo control plants from open field. No significant differences in nuclear DNA content were detected among plants of different origin. Chromosome counting in root tip meristems also showed normal tetraploid chromosome number (2n = 4x = 32) in ‘Cacanski Rubin’ shoots and normal triploid chromosome number (2n = 3x = 24) in Gisela 5 shoots regenerated in vitro. The results obtained suggest that no major genetic instability occurred during adventitious regeneration under the described experimental conditions.

Adventitious bud regeneration from the stigma of Sinapis alba L.

Stigmas isolated from flower buds of 'Nakielska' variety of &lt;i&gt;Sinapis alba&lt;/i&gt; were used to develop a micropropagation method suitable for breeding of new cultivars. The origin of adventitious bud regeneration was studied on MS medium, under stimulation by bezylaminopurine (BAP) in combination with 2,4-D - dichlorophenoxyacetic acid (2,4-D). Histological analysis showed the structure of Sinapis stigma (composed from four types of tissue: papillae, transmitting tissue, parenchyma and vascular bundles) and revealed that numerous meristematic centers developed from parenchyma cells in close vicinity of vascular bundles. Buds very quickly appeared on the surface of initial explants and later formed multiplantlets that were easily rooted in the soil.

Shoot multiplication and plant regeneration in Caragana fruticosa (Pall.) Besser

Different nutrient media can affect in vitro culturing protocols, and experimentation under varied growth conditions is valuable in plants where in vitro methods are in preliminary stages. We carried out the first in vitro propagation studies for the endangered species Caragana fruticosa (Fabaceae). We evaluated various nutrient media for their impact on shoot elongation and axillary bud proliferation using different concentrations of 6-benzylaminopurine (BA) and α-naphthaleneacetic acid (NAA). Shoot elongation was evaluated based on adventitious shoot primary culture and subculture regeneration from Caragana seedlings. Our goal was to improve both micropropagation and regeneration in C. fruticosa. MS nutrient media was superior to 1/2MS macronutrients, DKW, QL, and WPM for shoot elongation and axillary shoot proliferation. Shoots grown on 1/2MS and WPM exhibited some chlorosis, and shoots on QL produced larger leavers than plants growing on normal medium. The shoot proliferation coefficient on MS media supplemented with 2.22 μM BA and 0.44 μM BA + 2.69 μM NAA was significantly higher than that with other treatments in the primary culture. Shoots on 2.22 μM BA showed a higher proliferation coefficient (3.17) than others in the subculture. Shoots were rooted on 1/2MS medium with the addition of different concentrations of NAA. The optimal concentration for rooting was 0.27 μM NAA (74%). Roots exhibited many stout and long root hairs. Survivl of established plantlets was 82% at 30 days after transfer to soil. Plants established in the green house showed normal growth and displayed no apparent morphological differences compared to stock plants.

Expression of almond Knotted1 homologue (PdKn1) anticipates adventitious shoot initiation

The transcription factor encoded by the gene Knotted1 is a nuclear homeodomain protein, regulating meristematic cells at the shoot apical meristem. It has been demonstrated that Knotted1 (KN1) expression specifies stem cell fate in adventitious shoot induction in herbaceous plants. This gene may thus potentially identify the initiation of meristem development in adventitious shoot induction in difficult-to-regenerate plants such as Prunoideae. We isolated an almond (Prunus dulcis Mill.) KN1-type gene using degenerate primers targeting the most conserved regions of Knotted1 gene. The 5′ and 3′ ends of the isolated sequence were obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR), and the gene was named P. dulcis Knotted1-like (PdKn1). PdKn1 transcripts were detected by reverse transcription (RT)-PCR mainly in shoot apical and axillary meristems. The RT-PCR and RT-quantitative PCR PdKn1 expression in almond leaf explants was found to anticipate the organization of adventitious shoot meristems. Apricot RNA isolated from induced leaf explants cross-hybridized with the almond probe PdKn1 in Northern blotting. We suggest that almond PdKn1 may be a useful marker to study the adventitious regeneration system by revealing the potential organogenic conditions, not only in almond but also in other Prunoideae.

DETECTION OF SOMACLONAL VARIATIONS IN POTATO USING RAPD MARKERS

Potato (Solanum tuberosum L.) is an economically important vegetable crop in Egypt. It is the fourth most important crop by volume of production; it is high yielding, having a high nutritive value and gives high returns to farmers. Moreover, potato is considered a good source of antioxidants (Chen et al., 2007). It is vegetatively propagated, heterozygous and tetraploid, thus, traditional breeding of potato is very difficult (Solmon-Blackburn and Baker, 2001). Biotechnology could be contributed to solve this problem and realize great benefit to potato farmers. The regeneration of plants from cell and tissue culture represent an essential component of biotechnology and have the potential not only to improve the existing cultivars, but also for the generation of novel plants in a comparatively short time compared to conventional breeding. The success of plant biotechnology relies on several factors which include an efficient tissue culture system for regeneration of plants from cultured cells and tissues. During tissue culture, changes of interest to plant breeders may be heritable and result from changes in the plastid or nuclear genome. The introduction of variation may also be either problematic or useful for horticulturists and plant breeders and may occur in high frequency during adventitious plant regeneration or long-term callus culture (Kaeppler et al., 2000). Many researchers studied how to standardize the optimum concentrations of growth regulators for regeneration of potato and consequently great progress has been made in potato callus induction and plant regeneration (Ahloowalia, 1982; Dobranszki et al., 1999; Hansen et al., 1999; Ehsanpour and Jones, 2000; Fiegert et al., 2000; Yasmin et al., 2003; Shirin et al., 2007; Khadiga et al., 2009; Khalafalla et al., 2010; Shahabud-din et al., 2011). DNA markers provided valuable tools in various analyses ranging from phylogenetic to the positional cloning of genes. Scoring of changes morphological and biochemical in plant can be useful in some studies, but there is limited diversity and trait may be affected by environmental influences. Molecular techniques such as Random Amplified Polymorphic DNA (RAPD) is often favored over traditional phenotypic, cytological and biochemical analysis, and generally assess even small variations in the genome. Detection of somaclonal variations using RAPD markers has several advantages, since RAPD markers are technically simple, quick to perform with small amount of DNA and do not require previous information about genome or radioactive labeling (Michelmore et al., 1991). RAPDs are usually dominant and are inherited in a simple Mendelian fashion. Thus RAPD analysis is a useful tool in determining genetic relationships among regenerated potato and their original cultivars. The use of the PCR-based RAPD technique to detect somaclonal variations has been applied successfully to several plant species, such as Lolium (Wang et al., 1993), Allium sativum L (Al-Zahim et al., 1999) and Picea abies (Heinze and Schmidt, 1995). It has also been applied for tomato (Soniya et al., 2001) and potato (Khatab, 2000). The objectives of this study was to investigate the efficiency of callus induction and plant regeneration media for four potato cultivars and also to detect the somaclonal variations appeared after plant regeneration using RAPD markers.

Adventitious shoot regeneration from hypocotyl slices of mature apricot ( Prunus armeniaca L.) seeds: A feasible alternative for apricot genetic engineering

Adventitious shoot regeneration from hypocotyl slices of mature apricot seeds has been achieved with regeneration percentages of 31.7%, 44.4%, and 46.9% for the cultivars ‘Canino’, ‘Dorada’, and ‘Moniqui’, respectively. Regeneration was significantly affected by the parental origin of the explants ( P < 0.05) but not by thidiazuron or 3-indolebutyric acid for any of the three cultivars, at the levels tested. None of the other factors studied (basal medium, 2,4 dichlorophenoxy-acetic acid pulses, dark incubation period, or addition of silver thiosulfate) affected significantly shoot regeneration percentage from ‘Canino’ hypocotyl sections. The effect of paromomycin on regeneration was genotype-dependent and different dose–response curves were obtained for each cultivar. While 40 μM paromomycin completely inhibited regeneration from ‘Canino’ sections, some buds were obtained from ‘Dorada’ and ‘Moniqui’ explants. The two aminoglycoside antibiotics tested, kanamycin and paromomycin, showed differing toxicity on ‘Canino’. A lower concentration of kanamycin (20 μM) than of paromomycin inhibited totally adventitious regeneration from ‘Canino’ explants. Agrobacterium-mediated transformation experiments, with the non-oncogenic strain AGL1 harboring the binary plasmid p35SGUSINT, were performed and GUS assays were carried out after four weeks to determinate stable transformation events. The utilization of paromomycin (10 μM) as the selective agent increased significantly both the number of explants that presented at least one transformation event ( P < 0.05) and the number of large area or calli expressing the gus gene ( P < 0.001), compared with the addition of kanamycin (10 μM). Moreover, when 10 μM paromomycin was added to the medium some massively transformed explants were observed and a chimerical bud was regenerated.