Procedure For Plant Regeneration Research Articles

Somatic embryogenesis as potential method for commercial propagation in Passiflora edulis Sims f. edulis - an important horticultural crop

Purple passion fruit (Passiflora edulis Sims f. edulis) is one of the species with high commercial value in the genus Passiflora. In this study, the somatic embryogenesis (SEs) obtained from different explants (ex vitro leaf and in vitro root) cultured on medium-supplemented auxins and cytokinins, silver nanoparticles (AgNPs) enhanced SEs and plantlets derived from SEs were evaluated for the first time. The ex vitro leaf blade (1 × 1 cm) and in vitro root fragment (0.1 × 1 cm) were used as the initial explants. The results showed that the addition of auxins and cytokinins in culture medium stimulated SEs in both explant types. Root explant only induced callus on medium supplemented with 2,4-D (1.0 - 4.0 mg L−1), however, somatic embryos were directly induced with 1.0 mg L−1 NAA. While 1.0 - 2.0 mg L−1 2,4-D stimulated embryogenic callus induction (14.81 - 25.93%), NAA stimulated direct embryogenesis from leaf explants. The medium supplemented with 2.0 mg L−1 NAA combined with 1.5 mg L−1 TDZ significantly improved the SEs rate (81.48%) and number of embryos per explant (26.67 embryos) formed directly from leaf explants. On the other hand, the synergism between 2.0 mg L−1 2,4-D and 2.0 mg L−1 AgNPs significantly enhanced callus-derived embryogenesis from leaf explant with the highest SEs rate (92.59%) and number of embryos per explant (31.67 embryos). In addition, AgNPs also had a positive effect on promoting the maturation of callus-derived embryos. The plantlets derived from SEs were acclimatized in the greenhouse with a survival rate of over 90.00%. The SEs-derived plants flowered and produced fruit normally under field conditions. The results revealed a reliable plant regeneration procedure through direct or indirect SEs derived from ex vitro leaf and in vitro root explants and AgNPs-stimulated SEs that can be applied for commercial propagation of this crop.

Highly efficient heritable genome editing in wheat using an RNA virus and bypassing tissue culture

Genome editing provides novel strategies for improving plant traits but mostly relies on conventional plant genetic transformation and regeneration procedures, which can be inefficient. In this study, we have engineered a Barley stripe mosaic virus–based sgRNA delivery vector (BSMV-sg) that is effective in performing heritable genome editing in Cas9-transgenic wheat plants. Mutated progenies were present in the next generation at frequencies ranging from 12.9% to 100% in three different wheat varieties, and 53.8%–100% of mutants were virus free. We also achieved multiplex mutagenesis in progeny using a pool of BSMV-sg vectors harboring different sgRNAs. Furthermore, we devised a virus-induced transgene-free editing procedure to generate Cas9-free wheat mutants by crossing BSMV-infected Cas9-transgenic wheat pollen with wild-type wheat. Our study provides a robust, convenient, and tissue culture–free approach for genome editing in wheat through virus infection.

Effects of Phytohormone and Regulators on Shoot Tip and Nodal Explants for In Vitro Shoot and Root Clonal Propagation of Vitex negundo L.

Medicinal plants are one of the most vital natural resources, but many of them are currently endangered due to habitat loss. Consequently, it is critical to emphasize the importance of using micropropagation techniques for mass propagation of plantlets on a commercial scale, in addition to germplasm conservation and distribution. Nodal explants and shoot tips were expunged from 15 days of the explant by aseptic seedlings, an effective, quick, and better in vitro plant regeneration procedure for Vitex negundo L. has been developed. The recent study was considered to develop an in vitro procedure for the regeneration of V. negundo L., a traditional medicinal plant. Nodal segments and shoot tips were cultivated on MS medium enhanced with numerous plant growth regulators. For multiple shoots and root regeneration, various cytokinins were examined. 6-benzyl-aminopurin (BAP), kinetin (Kin), and 1H-indole-3-butanoic acid (IBA) were all tested as a supplement to Murashige and Skoog (MS) medium including auxin phytohormone, such as Indole acetic acid (IAA) and 1-naphthaleneacetic acid (NAA). The furthermost effective surface sterilization treatment for explants of V. negundo has been found 0.1% HgCl2 for 8 minutes. In all treatments, multiple shoots were collected from shoot tips and nodal segments. In MS media added with 2.0mg/l BAP, the most shoots were seen in V. negundo. Furthermore, V. negundo regeneration shoots rooted effectively in half MS containing 1.0 mg/l IBA. Finally, proliferated plantlets were effectively adapted in soil, where they grew normally without morphological anomalies and had a survival rate of 92 percent.

High-Efficiency Agrobacterium rhizogenes-Mediated Transgenic Hairy Root Induction of Lens culinaris

Previous efforts to transform lentil have been considerably hampered by the crop’s recalcitrant nature, giving rise to particularly low transformation and regeneration frequencies. This study aimed at optimizing an Agrobacterium rhizogenes-mediated transformation protocol for the generation of composite lentil plantlets, comprised of transgenic hairy roots and wild-type shoots. Transformation was performed by inoculating the cut hypocotyl of young lentil seedlings, while optimization involved the use of different bacterial strains, namely R1000, K599 and Arqua, and protocols differing in media composition with respect to the presence of acetosyringone and MES. Composite plantlets had a transgenic hairy root system characterized by an increased number of hairy roots at the hypocotyl proximal region, occasionally showing plagiotropic growth. Overall findings underline that transformation frequencies are subject to the bacterial strain, media composition as well as their combined effect. Among strains tested, R1000 proved to be the most capable of hairy root formation, while the presence of both acetosyringone and MES in inoculation and culture media yielded considerably higher transformation rates. The transgenic nature of hairy roots was demonstrated by the Ri T-DNA-mediated transfer of the rolB2 gene and the simultaneous absence of the virCD sequence of A. rhizogenes. Our findings provide strong evidence that A. rhizogenes-mediated transformation may be employed as a suitable approach for generating composite seedlings in lentil, a species whose recalcitrance severely hampers all efforts addressed to transformation and whole plant regeneration procedures. To the best of our knowledge, this is the first report on the development of a non-laborious and time-efficient protocol for the generation of transgenic hairy roots in lentil, thus providing an amenable platform for root biology and gene expression studies in the context of improving traits related to biotic and abiotic stress tolerance.

Evaluation of Operational Cycles for Long‐Term Run of a Tar Removal Catalytic System

AbstractBiomass gasification experiments on pilot/demo scales have some issues related to the early deactivation of catalysts during the tar removal step. To avoid this problem, a method was developed in a bench‐scale micro activity unit using toluene as tar model compound in order to suppress this effect. The runs were performed with a commercial Pt catalyst supported on Ce‐Zr‐Al, alternating periods of regeneration and reactivation steps with steam, nitrogen, and hydrogen. The toluene steam reforming using operational cycles in order to reach a long‐term run provided useful information for pilot plant studies, mainly reactivation and regeneration procedures. The main concern on tar removal studies by steam reforming is the catalyst deactivation due to the presence of polyaromatic and olefinic compounds on the material pores, which is produced during biomass gasification.

Functional\xa0characterization of the PHT1 family transporters of foxtail millet with development of a novel Agrobacterium-mediated transformation procedure

Phosphate is an essential nutrient for plant growth and is acquired from the environment and distributed within the plant in part through the action of phosphate transporters of the PHT1 family. Foxtail millet (Setaria italica) is an orphan crop essential to the food security of many small farmers in Asia and Africa and is a model system for other millets. A novel Agrobacterium-mediated transformation and direct plant regeneration procedure was developed from shoot apex explants and used to downregulate expression of 3 members of the PHT1 phosphate transporter family SiPHT1;2 SiPHT1;3 and SiPHT1;4. Transformants were recovered with close to 10% efficiency. The downregulation of individual transporters was confirmed by RT-PCR. Downregulation of individual transporters significantly reduced the total and inorganic P contents in shoot and root tissues and increased the number of lateral roots and root hairs showing they have non-redundant roles. Downregulation of SiPHT1;2 had the strongest effect on total and inorganic P in shoot and root tissues. Complementation experiments in S. cerevisiae provide evidence for the ability of SiPHT1;1, 1;2, 1;3, 1;7 and 1;8 to function as high affinity Pi transporters. This work will aid development of improved millet varieties for global food security.

Validation of reference genes for qPCR analysis of Coffea arabica L. somatic embryogenesis-related tissues

Quantitative real-time PCR (qPCR) is a sensitive method used to investigate relevant changes in gene expression during somatic embryogenesis. Understanding its regulatory network might be helpful to the process of induction of embryos and facilitate the development of efficient plant regeneration procedures. In this study, a set of 12 genes was selected and their stability was assessed in different tissues of somatic embryogenesis-related cultures of Coffea arabica. Analyses of gene expression stability were performed using the RefFinder tool that integrates the geNorm, NormFinder, BestKeeper and Delta-Ct algorithms. Among the all candidate reference genes studied, APRT/EF1a, UBQ/ACT, ACT/24S, RPL39/24S, PP2A/RPL39, PP2A/AP47, emerged as the most stable for normalization of qPCR analyses of embryogenic cell suspensions, non-embryogenic calli, embryogenic calli, combined embryogenic and non-embryogenic calli, somatic embryos and plantlet, respectively. A combination of two genes, 24S and PP2A, was identified as most suitable reference genes across all samples for the C. arabica tissues studied. The commonly employed reference gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was found to be inappropriate as a reference gene for embryogenic tissues of C. arabica. In addition, Baby boom (BBM) gene expression was investigated to confirm the validity of the selected reference genes, the transcript levels of gene were overestimated when unsuitable reference genes were used for normalization. The results shown herein will permit a more precise and reliable normalization of qPCR in experiments involving somatic embryogenesis of C. arabica.

Induksi Kalus dan Regenerasi Beberapa Genotipe Gandum (Triticum aestivum L.) secara In Vitro

<p>Callus Induction and In Vitro Plant Regeneration of<br />Wheat Genotypes (Triticum aestivum L.). Atmitri<br />Sisharmini, Aniversari Apriana, and Sustiprijatno. Development<br />of a reliable in vitro plant regeneration procedure for<br />wheat is a prerequisite for its improvement by genetic transformation.<br />The purpose of this study was to obtain methods<br />of callus induction and regeneration of wheat genotypes.<br />This experiment was conducted at ICABIOGRAD. Immature<br />embryos from four wheat genotypes, ie Perdix, Naxos Wew,<br />Combi and Fasan were used to induce callus formation and<br />regeneration rate of callus. For the preparation of callus<br />induction medium, MS-L7 basal medium was supplemented<br />with combination of growth regulators 2,4 dichlorophenoxy<br />acetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid<br />(picloram). While, plant regeneration medium was prepared<br />using MS basal medium supplemented with combination of<br />three growth regulators i.e. IAA, BAP and kinetin. The results<br />showed that genotype, in vitro culture medium and growth<br />regulators played a dominant role in callus induction and<br />plantlet regeneration. All the 4 genotypes responded positively<br />to callus induction, however, variability was observed<br />not only among the genotypes but also within callus<br />induction medium used. The best induction medium was<br />the MS-L7 basal medium supplemented with combination of<br />phytohormon 4 mg/l 2,4-D + 2 mg/l picloram (GIK-3) which<br />showed 100% callus induction frequency. Whereas, the best<br />regeneration medium was shown by MS basal medium with<br />combination of phytohormon 1.5 mg/l BAP dan 0.5 mg/l<br />kinetin (RG3). Regarding plant regeneration, Perdix was the<br />most responsive genotype to be regenerated with regeneration<br />frequency of 57.33%. The successfully acclimatized<br />planlets in greenhouse were obtained from Perdix and<br />Naxos Wew genotypes. These results will potentially facilitate<br />genetic transformation research of wheat in Indonesia.</p>

In Vitro Propagation of Camellia oleifera Abel. Using Hypocotyl, Cotyledonary Node, and Radicle Explants

Camellia oleifera Abel. is one of four major woody oil plants in the world. The objective of the current study was to evaluate the effect of different plant growth regulators (PGRs) and concentrations on direct organogenesis using cotyledonary nodes, hypocotyls, and radicle explants. High induction frequency of adventitious shoots were obtained from cotyledonary nodes, hypocotyls, and radicle explants (85.2%, 73.6%, and 41.0%, respectively) when cultured on half-strength Murashige and Skoog (1/2 MS) medium containing 2.0 mg·L−1 6-benzylaminopurine (BA) and 0.1 mg·L−1 indole-3-acetic acid (IAA). Microshoots from cotyledonary nodes, hypocotyls, and radicle explants were then transferred to 1/2 MS medium containing 2.0 mg·L−1 BA and 0.05 mg·L−1 indole-3-butyric acid (IBA) for shoot multiplication, resulting in 6.9 shoots per explant. The shoots were transferred to Woody Plant Medium (WPM) supplemented with various α-naphthalene acetic acid (NAA) and gibberellic acid (GA3) for shoot elongation. The mean length of shoots and the number of leaves per shoot were 3.7 and 6.6 cm, respectively, in WPM supplemented with 0.5 mg·L−1 NAA and 3.0 mg·L−1 GA3. The highest rooting of shoots (90.2%) or the number of roots per shoot (7.2) was obtained when elongated microshoots were transferred to 1/2 MS medium supplemented with 3.5% perlite, 1.0 mg·L−1 IBA and 2.0 mg·L−1 NAA. The rooted plantlets were successfully acclimatized in the greenhouse with a survival rate of 90.0%. The in vitro plant regeneration procedure described in this study is beneficial for mass propagation and improvement of C. oleifera through genetic engineering.

Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423

Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4–5 times subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological analysis and scanning electron micrographs confirmed the different developmental stages of somatic embryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somatic embryogenesis. Differentially expressed genes were cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regeneration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA sequences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423.Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somatic embryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize.

Agrobacterium tumefaciens-mediated transformation of axillary bud callus of Hibiscus rosa-sinensis L. ‘Ruby’ and regeneration of transgenic plants

Hibiscus rosa-sinensis L. is a popular ornamental species valued for its large brightly coloured ephemeral flowers and has a range of health-promoting properties. The value of H. rosa-sinensis could be improved even further if there were ways to prolong the display life of its short-lived flowers, and to improve its frost tolerance. Development of an efficient plant transformation and regeneration procedure that allows introduction of genes into the plant will greatly facilitate this. Here we outline a transformation and regeneration procedure that is the first to produce transformed H. rosa-sinensis plants successfully. We first optimised callus induction and shoot regeneration efficiency. The highest shoot regeneration frequency of 66.7 % was achieved in the cultivar ‘Ruby’ when callus induced from axillary buds using a basal medium supplemented with 2.22 µM benzylaminopurine and 2.47 µM β-naphthoxyacetic acid was cultured on shoot regeneration medium. The frequency of shoot regeneration from callus was lower in ‘Ben James’ and absent in ‘Bright Light’, indicating genotypic differences. When axillary bud-derived callus of ‘Ruby’ was co-cultured with Agrobacterium tumefaciens harbouring a β-glucuronidase (GUS) reporter plasmid, 49 % of calli produced shoots on selection media. All tested plantlets were confirmed as transformed based on the presence of the GUS transgene in the genomic DNA and GUS activity measurements. Roots were induced on transgenic plantlets using half-strength basal medium supplemented with 2.85 µM indole-3-acetic acid. This simple protocol can be used to improve the ornamental, agronomic and health-promoting traits of H. rosa-sinensis hitherto recalcitrant to A. tumefaciens-mediated transformation.

Effect of Plant Growth Regulators onin vitroCallus Induction and Plant Regeneration from Mature Wheat(Triticum aestivumL.) Embryos

Genetic improvement of hexaploid bread wheat (Triticum aestivum L.) through transfor-mation technology is based upon a simple, effi-cient and reliable in vitro plant regeneration pro-cedure. In the present study, we have reported the effects of three plant growth regulators, 2, 4-Diphenoxyacetic acid (2, 4-D), Benzyl adenine (BA) and Indole acetic acid (IAA) on in vitro callus induction and plant regeneration from mature embryos in three commercial bread wheat geno-types viz., Raj3765, K7903 and K9533. High fre-quency callus induction and plant regeneration was observed in all the three tested wheat geno-types on basal MS media fortified with 2, 4-D, BA and IAA at different concentrations. Among the three tested wheat genotypes in the current study, Raj3765 showed best results both in terms of callus induction and plant regeneration from mature embryos. The effects of 2, 4-D, BA and IAA on callus induction and plant regeneration were genotype dependent. The procedure thus developed would facilitate the improvement of agronomically desirable traits in wheat geno-types through genetic transformations.

Agrobacterium tumefaciens-mediated genetic transformation of Digitalis purpurea L.

Genetic transformation is a tool of special interest for developing new biotechnological strategies for the production of bio-active compounds such as cardenolides, which are exclusively obtained from plants. To date, Digitalis plants are the main economically viable source of cardenolides for the pharmaceutical industry. This study describes the development of efficient plant regeneration and Agrobacterium-mediated genetic transformation protocols for Digitalis purpurea L. First, a plant regeneration procedure starting from leaf segments of in vitro-cultivated plants was established and the minimal inhibitory concentration of G-418 (geneticin) for callus induction was determined. Both leaf segments and callus tissue were sensitive to G-418 70 mg l−1. Afterwards, two Agrobacterium strains were used to test their T-DNA transfer ability on D. purpurea leaf tissues, EHA105 and C58C1RifR (pMP90), both harboring the binary vector pTJK136. Strain C58C1RifR (pMP90) yielded a higher number of transformed plants than EHA105. Successful transformation was confirmed by histochemical β-glucuronidase (GUS) assays of the putative transgenic tissues and PCR analyses using β-glucuronidase (uidA)- and neomycin phosphotransferase II (nptII)-specific primers. Southern blot hybridization confirmed the stable integration of the nptII gene in the transgenic plants. In total, 518 independent transgenic lines were regenerated with an average of 6.91 transgenic lines per initial leaf segment infected with A. tumefaciens strain C58C1RifR (pMP90). To date, only a few studies have been published on the genetic transformation of Digitalis species. The protocols for plant regeneration and genetic transformation described in this paper will contribute to functional studies for a better understanding of cardenolide biosynthetic pathways and the metabolic engineering of cardenolides to develop high-yielding improved genotypes.

Transformation of tef (Eragrostis tef) by Agrobacterium through immature embryo regeneration system for inducing semi-dwarfism

Successful application of genetic transformation for integration of a transgene is much dependent upon availability of an efficient in vitro plant regeneration procedure and detection of transgene insertion and expression. Isolated immature embryos (IEs) of Eragrostis tef cultivar DZ-01-196 were used for embryogenic callus formation and the callus was transformed with GA inactivating gene PcGA2ox under the control of a triple CaMV 35S promoter using Agrobacterium transformation procedure. Embryogenic callus was induced from immature embryos in a medium containing KBP minerals in the presence of 2,4-dichlorophenoxiyacetic acid. The embryogenic calli were further inoculated with Agrobacterium and the calli were grown in co-cultivation medium (CCM) followed by selection in KBP and regeneration (K4NB) media. Putatively transformed E. tef embryogenic calli were tolerant to treatment with the selectable marker kanamycin, while 75mgl−1 geneticin inhibited growth of non-transformed shoots derived from matured embryos completely after 12days. A total of 55 plants were regenerated from all the embryogenic calli to fully viable plants setting seeds at maturity. Eight putatively transformed T0 plants were produced carrying the transgene in their genome which was detected by PCR. Sequence analysis confirmed amplified PCR products to have 97.2 and 99.8% sequence identity to PcGA2ox and nptII, respectively. However, detection of the transgene, PcGA2ox or nptII, in T1 plants was inconsistent although phenotypic analysis of T1 plants showed changes in pheno-morphic and agronomic characters such as plant height, number of internodes, tillering, panicle length, biomass, yield as well as GA content. Culm reduction was due to absence of elongation of the upper-most internodes. Panicle length in semi-dwarfed plants showed no relation with culm length. GA analysis showed plants with semi-dwarf phenotype to be associated with a low level of bioactive GA1 and its immediate precursors. Up to 3.7 fold increase in grain yield per plant was found in some semi-dwarfed plants.

Epigenesis ThroughIn-vitroRegeneration in Soybean Amenable to Genetic Transformation

An efficient and reproducible plant regeneration procedure is essential for introducing genes of interest in important crops through genetic transformation. However, some crops, such as soybean [Glycine max (L.) Merrill), are difficult to manipulate in vitro, often depending on their genotype, and the reproduction of the established protocols is not always possible. The purpose of this paper is the optimization of a regeneration protocol for soybean shoots of the Indian variety JS-335 to enable its reproduction. Four explants viz, Cotyledonary node, embryo, half seed and hypocotyl were tested using various combinations and concentrations of growth hormones. The effect of several concentrations of 2,4-D and BAP was effective in initiating Callogenesis in three types of explants though concentration varied widely. The highest callogenesis of 82.93% were recorded on 14th day using half seed explant. However, the highest shoot induction of 93.79% with multiple numbers of shoots was recorded using cotyledonary node explant inoculated on the medium fortified with 5.0 mg.L−1 BAP.

In vitro plant regeneration from unpollinated ovaries of Allium chinense

The present work was conducted to develop a simple and efficient protocol for in vitro shoot regeneration and plantlet formation from unpollinated ovary culture in Allium chinense. Ovaries that were excised from flowers 2–3 days before anthesis and cultured on induction medium with 9.05μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 8.88μM benzylaminopurine (BAP) for 2 weeks gave the best organogenic response (56.32%). For shoot regeneration, the highest percent organogenic ovaries (64.53%) were obtained on B5 induction medium containing 9.05μM 2,4-D and 8.88μM BAP. Ovaries cultured on B5 containing 10.74μM naphtaleneacetic acid (NAA) and 0.44μM BAP exhibited maximum shoot regeneration (60.32%). Shoots elongated and produced normal plantlets when cultured on B5 medium supplemented with 10.74μM NAA for 2 weeks. The plant regeneration procedure from the ovary may be an alternative for the improvement of A. chinense by genetic engineering.

The specificity and genetic background of the rye (Secale cereale L.) tissue culture response

Rye is one of the most important crops in Eastern and Northern Europe. Despite the numerous beneficial features of rye, its annual production decreases successively which correlates with the lack of progress in its breeding compared with other cereals. Biotechnological methods could effectively improve the breeding of rye. However, their application is highly limited by the absence of an efficient procedure for plant regeneration in vitro, since rye is one of the most recalcitrant cereals with regard to the tissue culture response (TCR), and successful regeneration is highly dependent on genotype. Efforts to understand the genetic mechanisms controlling TCR of rye have elucidated some basic aspects, and several genes and genome regions controlling this trait have been identified. The aim of this review is to summarize the limited current knowledge of this topic.

Effects of different combinations of benzyl adenine and indole acetic acid concentrations on in vitro plant regeneration in hexaploid wheat

Development of a reliable in vitro plant regeneration procedure for hexaploid bread wheat (Triticum aestivum L.) is a prerequisite for its improvement by genetic transformation. Here, we report the effects of two growth regulators, benzyl adenine (BA) and indole acetic acid (IAA) on callus induction and plant regeneration from scutellum cultures of two commercial bread wheat cultivars: Giza 164 and Sakha 69. Callus induction was obtained from isolated embryos cultured on modified Murashige and Skoog (MS) basal medium. After four weeks of callus induction, all calli were plated on MS basal medium for regeneration. Wheat genotype and callus induction medium played a dominant role in plantlet regeneration. 2.0 mg/L BA and 0.2 mg/L IAA were the best combinations for inducing callus and let to highest regeneration frequency (81.67%) across the cultivars. Overall, based on our medium conditions, Giza 164 displayed higher regeneration frequency (81.11%) than Sakha 69. These results will facilitate genetic transformation for the economic varieties Giza 164 and Sakha 69.

Establishment of an efficient callus induction and plant regeneration system in some wheat (Triticum aestivum L.) cultivars grown in Sudan

A procedure for callus induction and plant regeneration was developed for wheat ( Triticum aestivum ) cultivars grown in Sudan. Mature embryos of cultivar Khaleefa removed from surface sterilized seeds were used as explants. For callus induction, explants were cultured on Murashige and Skoog medium (MS) supplemented with different levels (1.5 to 4.0 mg/l) of 2,4-dichlorophenoxy acetic acid (2,4-D), indole-3- butyric acid (IBA) and indole-3- acetic acid (IAA). After 4 weeks of culture, the highest callus fresh weight (0.05 ± 0.01 g) was obtained on MS medium supplemented with 2.0 mg/L of 2,4-D. For shoot regeneration, the developed calli were transferred to MS medium without growth regulators or with different levels (0.1 to 3.0 mg/l) of 5-phenylcarbamoylamino-1,2,3-thiadiazole (thidiazuron) (TDZ), benzyladenine (BA) and kinetin (KN) alone or in combination with 2.0 mg/L of 2,4-D. After 6 weeks of culture, the best result for regeneration percentage (40%) was obtained on MS medium without growth regulators. Callus derived shoots were rooted most effectively in half-strength MS medium without or with different levels of IBA. To determine the genotypic effect, the developed procedure was used to evaluate the regeneration ability of other 5 Sudanese wheat cultivars. Significant differences were observed between genotypes for plant regeneration ability, indicating that these criteria are genotype dependent. The acclimatized regenerated plants were morphologically uniform with normal leaf shape and growth pattern under greenhouse conditions. Key words : Triticum aestivum , callus induction, regeneration, 2,4-dichlorophenoxyacetic acid (2,4-D), genotypes.

EFFECT OF AUXIN-CYTOKININ INTERACTION AND LIGHT CONDITIONS ON ORGANOGENESIS IN MATURE LEAF EXPLANTS OF PELARGONIUM RAPACEUM (L.) L¿HÉRIT

Pelargonium rapaceum (L.) L’Herit. belongs to the family Geraniaceae, and is classified in section Hoarea within the genus Pelargonium. In general, it propagates through seed and tuber division which produces a small number of stock plants. Alternative technology can be used for mass in vitro propagation which has the potential to benefit from large scale propagation of pathogen indexed plants including rapid multiplication. The objectives of this work were to create efficient plant regeneration procedures in P. rapaceum using mature leaf explants. Leaves were cultured on MS medium supplemented with different concentrations of auxin at 0, 0.1, 1 and 5 mg/L of NAA or IAA used in combination with cytokinin at 0, 0.1, 1 and 5 mg/L of BAP or TDZ. Explants were cultivated under a white fluorescent lamp for 16 h (20-30 µmol/s/m2) or dark conditions in a culture room at 24°C. Significant differences were observed in shoot organogenesis under the different concentrations of growth regulator and culture conditions. Morphogenesis occurred in response to the growth regulator combinations tested. The initial growth occurred after 1 to 2 weeks of culture. The 100% regeneration was found for explants cultured on medium supplemented with a combination of NAA with BAP, and IAA with TDZ. No shoot was observed in any medium containing TDZ. Shoots occurred with explants cultivated on MS medium containing NAA in combination with BAP, and IAA in combination with BAP. Light incubation was needed for explants cultivated on medium using combinations of IAA while the cultures on medium using combinations of NAA resulted in shoot regeneration under both culture conditions. However, the highest number of shoots was obtained from explants cultivated under darkness. Percentage of shoot regeneration and the number of shoots per explant were also observed and discussed.