Shoot Regeneration Medium Research Articles

In vitro-regenerated wetland sedge Eriophorum vaginatum L. is genetically stable

Eriophorum vaginatum L. is a promising species for phytostabilization, restoration, or creation of wetlands, because it can survive in cold, nutrient-poor, or metal-contaminated soils. However, its propagation on a large scale is problematic due to the infrequent production of viable seeds, seed dormancy, and the limitations of reproduction by rhizomes. A technique to rapidly and effectively produce large quantities of outplanting stock of this species was sought. Seeds of E. vaginatum were cultured on Murashige and Skoog (MS) medium supplemented with plant growth regulators at different concentrations. The highest regeneration rate was obtained on MS medium supplemented with 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.32 μM kinetin (KIN) for callus induction, and 17.76 μM BA (6-benzylaminopurine) for shoot regeneration as well as when 2.26 μM 2,4-D and 4.65 μM KIN was added to the callus-induction medium, and 8.88 μM or 17.76 μM BA to the shoot-regeneration medium. The regenerated shoots were rooted on MS medium without growth regulators and acclimatized in a greenhouse. Genetic stability of the in vitro regenerants was determined using flow cytometry and random amplified polymorphic DNA. Cytometric analysis revealed that the nuclear DNA content was similar in all plant materials and amounted to about 0.8 pg/2C. The PCR amplification products were monomorphic in callus-derived plants and similar to plants grown in a field. Lack of genome size variation and polymorphism within the regenerants indicates that the detailed E. vaginatum micropropagation protocol allows the production of a large number of genetically stable plants.

Identifying somaclonal variants of the apple rootstock Malling 7 resistant to white root rot

A study was conducted with the objective to develop a technique for in vitro screening of cells/shoots of apple rootstock Malling 7 (M7) against Dematophora necatrix culture filtrate. Large amounts of profuse, creamish white and friable calli was obtained from leaf explants when cultured on MS medium supplemented with 5mg/l naphthalene acetic acid (NAA) and 1mg/l Benzyladenine (BA). Adventitious shoots were regenerated from leaves cultured on MS medium containing BA (4.0, 4.5mg/l) or thidiazuran (TDZ, 0.2–1.0mg/l) and indole-3-acetic acid (IAA), indole butyric acid (IBA) or NAA (0.5–1.5mg/l) for 6 weeks. Friable callus as well as regenerants were exposed separately to selection medium fortified with different levels (10–90%) of fungal culture filtrate (FCF) of D. necatrix to isolate tolerant cells/regenerants. 5.18% callus lines and 40% shoots survived on 70% FCF which were further subjected to three cycles of treatment using continuous in vitro selection approach. The surviving calli were subcultured on shoot regeneration medium to obtain plantlets but were unable to regenerate. This study also examined total sugars, phenol, protein content and activity of enzyme polyphenol oxidase (PPO) in FCF tolerant calli, and found higher levels of all except sugars. FCF tolerant and unselected regenerants were rooted and hardened. These plants were then screened for disease tolerance by planting in pathogen infested soil in pots. The data revealed that all five regenerants survived over control after 60 days of inoculation, thus showing resistance to white root rot disease.

In vitro propagation and characterization of phenolic content along with antioxidant and antimicrobial activities of Cichorium pumilum Jacq.

Cichorium pumilum, a member of Asteraceae, is widely used as a traditional medicinal herb. An efficient protocol for callus formation and whole plant propagation has been developed. Callus cultures were induced from leaf explants on Murashige and Skoog (MS) medium supplemented with 1.5 mg l−1 6-Benzyladenine (BA) and 0.5 mg l−1 Naphthalene acetic acid (NAA). Maximum numbers of shoots were obtained from calli transferred to shoot regeneration medium containing MS basal medium with 1.5 mg l−1 BA or Kinetin (Kin). The shoots were effectively rooted on MS medium supplemented with different concentrations of Indole-3-butyric acid. In the present study, the antibacterial activity of C. pumilum extracts was assayed in vitro by agar disc diffusion and agar well diffusion methods against 10 different bacterial species. The results showed effect on the growth of 50 and 70% of the tested bacterial species using methanol and ethanol extracts respectively. Klebsiella pneumoniae was susceptible to the ethanolic and methanolic extracts of wild plants and in vitro tissues, whereas Enterococcus faecalis was resistant to all the extracts. This study verified that the methanol extracts have strong antioxidant activities with high levels of phenolic compounds. The antioxidant activity and total phenol content of callus cultures and in vitro plantlets were lower than those of the wild plants. The results obtained confirm the therapeutic potency of Cichorium used in the traditional medicine, in addition, the efficient in vitro production system developed in this study provide sterile and consistent tissues for the investigation of phytochemical and pharmacological effects and germplasm conservation of C. pumilum.

Propagation of goldenrod (Solidago canadensis L.) from leaf and nodal explants

Goldenrod (<em>Solidago canadensis </em>L.) is an invasive plant species in many countries except North America but a cut-flower species worldwide. There is a need to generate and propagate goldenrod clones efficiently for research and commercial purposes. A callus induction and plantlet regeneration system was developed by studying the influence of explant type and different concentrations of plant growth regulators. The highest callus production from leaf segments was obtained on Murashige and Skoog’s medium (MS medium) supplemented with 1.0 mg/L naphthalene acetic acid (NAA) and 1.0 mg/L 6-benzylaminopurine (BA). Adventitious shoots could be regenerated directly from leaf explants without an intermediate callus phase with the highest shoot induction percentage of 87.2%. The largest number of adventitious shoots per leaf explant (3.2) was obtained on MS medium supplemented with 0.4 mg/L NAA and 2.0 mg/L BA. MS medium supplemented with 0.1 mg/L NAA and 1.0 mg/L BA was the best medium for axillary shoot regeneration from nodal segments. The highest root number and longest roots occurred on half-strength MS without the addition of any growth regulator. Rooted plantlets were then transferred to a soil-based growth medium, placed in a greenhouse, and acclimatized with 100% success. All surviving plants grew normally without showing any morphological varia­tion when compared to those grow from seed. This regeneration protocol may be used to produce certain biotypes of goldenrod suitable for genetic transformation rapid propagation of goldenrod for commercial purposes or for screening fungi and toxins as potential biocontrol agents against this weed.

In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Parkia timoriana (DC.) Merr.: a multipurpose tree legume

In vitro regeneration of Parkia timoriana (DC.) Merr. has been achieved using cotyledonary node explants. The ability to produce multiple shoots has been evaluated using semi-solid Murashige and Skoog (MS) basal medium and Gamborg’s B-5 basal medium supplemented with various concentrations of α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BA) either in single or in combinations. The explants cultured in MS medium supplemented with combinations of 2.7 μM NAA and 11 μM BA showed the maximum frequency of multiple shoots (96.66%) formation and number of shoots per explants (6.60), respectively. For rooting, full and half strength MS medium supplemented with various concentrations of indole-3-butyric acid (IBA) and NAA were studied and the highest number of root formation was observed in full-strength MS supplemented with 9.8 μM IBA. Using Agrobacterium tumefaciens strain EHA105 pCAMBIA2301 various optimum conditions for efficient transformation were determined by recording the percentage of GUS+ explants. Following the optimized conditions, the co-cultured explants were cultured on semi-solid shoot regeneration medium containing MS medium + 2.7 μM NAA + 11 μM BA + 100 mg/l kanamycin + 500 mg/l cefotaxime. After 8 weeks of culture, the regenerated shoots were rooted in rooting medium (RM) containing MS medium + 9.8 μM indole-3-butyric acid (IBA), 3% sucrose, 7.5 mg/l kanamycin and 500 mg/l cefotaxime. Successful transformation was confirmed by histochemical GUS activity of the regenerated shoots, nptII gene PCR analyses of the regenerated kanamycin resistant plantlets and Southern analysis of putative transgenic PCR+ plants.

In vitro plant regeneration from cotyledonary nodes of recombinant inbred lines of lentil

An efficient and reproducible in vitro regeneration protocol for lentil was developed. For shoot regeneration, cotyledonary node explants of ten elite genotypes were cultured in an inverted orientation on different shoot regeneration media that consisted of Murashige and Skoog (MS) medium supplemented with 1 mg L −1 6-benzylaminopurine (BAP) (M1), 1 mg L −1 BAP + 0.45 mg L −1 indole-3-acetic acid (IAA) (M2), and 2 mg L −1 BAP (M3). High percentages of shoot regeneration ranging from 80 to 100% on M1 and M3 media and from 50 to 100% on M2 medium were induced. M1 was the most efficient shoot regeneration medium for most genotypes tested. For rooting, in vitro and in vitro– in vivo methods were used. Low and variable rooting percentages ranging from 0 to 45% were recorded with in vitro– in vivo method. Efficiency of rooting on in vitro medium varied depending on the medium in which shoots had been previously regenerated and the genotype tested. When M1 medium was used, high rooting percentages (over 40%) for most genotypes except for microsperma genotypes were found. When the 10 genotypes were screened for good regeneration performance using M1 medium, 2 main clusters and 3 subgroups within one of the clusters were formed based on similarities respect of the number of regenerated shoots per explant and rooting percentages. Subgroup 1 composed by A1146 genotype produced the highest number of shoots per explant (6.17 shoots) and a high rooting percentage (60%) so was selected for further transformation and use as a potential commercial variety.

Somatic hybrid plants of Nicotiana × sanderae (+) N. debneyi with fungal resistance to Peronospora tabacina

The genus Nicotiana includes diploid and tetraploid species, with complementary ecological, agronomic and commercial characteristics. The species are of economic value for tobacco, as ornamentals, and for secondary plant-product biosynthesis. They show substantial differences in disease resistance because of their range of secondary products. In the last decade, sexual hybridization and transgenic technologies have tended to eclipse protoplast fusion for gene transfer. Somatic hybridization was exploited in the present investigation to generate a new hybrid combination involving two sexually incompatible tetraploid species. The somatic hybrid plants were characterized using molecular, molecular cytogenetic and phenotypic approaches. Mesophyll protoplasts of the wild fungus-resistant species N. debneyi (2n = 4x = 48) were electrofused with those of the ornamental interspecific sexual hybrid N. × sanderae (2n = 2x = 18). From 1570 protoplast-derived cell colonies selected manually in five experiments, 580 tissues were sub-cultured to shoot regeneration medium. Regenerated plants were transferred to the glasshouse and screened for their morphology, chromosomal composition and disease resistance. Eighty-nine regenerated plants flowered; five were confirmed as somatic hybrids by their intermediate morphology compared with parental plants, cytological constitution and DNA-marker analysis. Somatic hybrid plants had chromosome complements of 60 or 62. Chromosomes were identified to parental genomes by genomic in situ hybridization and included all 18 chromosomes from N. × sanderae, and 42 or 44 chromosomes from N. debneyi. Four or six chromosomes of one ancestral genome of N. debneyi were eliminated during culture of electrofusion-treated protoplasts and plant regeneration. Both chloroplasts and mitochondria of the somatic hybrid plants were probably derived from N. debneyi. All somatic hybrid plants were fertile. In contrast to parental plants of N. × sanderae, the seed progeny of somatic hybrid plants were resistant to infection by Peronospora tabacina, a trait introgressed from the wild parent, N. debneyi. Sexual incompatibility between N. × sanderae and N. debneyi was circumvented by somatic hybridization involving protoplast fusion. Asymmetrical nuclear hybridity was seen in the hybrids with loss of chromosomes, although importantly, somatic hybrids were fertile and stable. Expression of fungal resistance makes these somatic hybrids extremely valuable germplasm in future breeding programmes in ornamental tobacco.

Induction of hairy roots and plant regeneration from the medicinal plant Pogostemon Cablin

An efficient transformation system for the medicinal and aromatic plant, Pogostemon cablin Benth was developed by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots formed directly from the cut edges of leaf explants or via callus stage 8 days after inoculation with the bacterium. The highest frequency of leaf explant transformation by Agrobacterium rhizogenes ATCC15834 was about 80% after infection for 25 days. Hairy roots grew rapidly on plant growth regulators (PGRs)-free Murashige and Skoog (MS) or 6,7-V medium and had characteristics of transformed roots such as fast growth and high lateral branching. The PCR amplification showed that rol genes of Ri plasmid of A. rhizogenes were integrated and expressed into the genome of transformed hairy roots. The hairy root line, PL6, grew very slowly in the first 8 days, then grew very quickly between day 8 and day 24. The optimum medium for callus induction of hairy roots consisted of 2.0 mg l−1 benzyladenine (BA) and 0.1 mg/l α-naphthaleneacetic acid (NAA); while optimum medium for adventitious shoot regeneration from these cultures consisted of 0.1 mg l−1 BA and 0.1 mg l−1 NAA. Adventitious shoots could be rooted on 1/2MS. Southern blot analysis confirmed that rol genes of TL-DNA of Ri plasmid was integrated with at least three copies into the genome of hairy roots- regenerated P. cablin plants. The results presented provide a solid foundation for production of patchouli essential oil from hairy roots or its regenerated plants and also provide possibilities for utilization of artifical polyploidization or chemical mutation of hairy roots for improving germplasm and breeding of a new cultivar of P. cablin.

Manipulation of ploidy for kiwifruit breeding: in vitro chromosome doubling in diploid Actinidia chinensis Planch.

Tetraploid plants were induced by colchicine treatment of in vitro leaf petiole segment cultures of five diploid Actinidia chinensis Planch. genotypes, including the commercially important, yellow-fleshed cultivar ‘Hort16A’, three female selections with red-fleshed fruit and one male pollinizer. Petiole segments were incubated on a shoot regeneration medium for a period of 4 weeks, and subsequently microshoots were treated with 0.05 or 0.1% colchicine. About one-third of the regenerated shoots were tetraploid following 0.05% colchicine treatment, more than with 0.1% colchicine treatment. Similar rates of tetraploid induction were achieved with all the genotypes tested. The efficiency of induction of polyploidy depended on the interaction between the types of in vitro culture chosen and the concentration of colchicine used. There are no previous reports of colchicine being used so successfully to induce polyploidy in Actinidia.

Establishment of regeneration and transformation system of sugarcane cultivar GT54-9 (C9)

Plant regeneration protocols for sugarcane GT54-9(C9) cultivar were developed for direct organogenesis and indirect somatic embryogenesis, using young leaf segments as explants by studying the influence of different concentrations and types of cytokinin and auxin hormones. For the callus formation from young leaves, a medium containing 4mg/l 2,4-D was found very effective. For embryo formation, MS medium supplemented with 1mg/l Kin and 0.5 mg/l 2,4-D was used. While in the case of direct organogenesis protocol, the medium containing 1mg/l BAP and 2mg/l NAA was the best for direct shoot formation. Data showed that the best shoot regeneration and elongation medium for direct organogenesis and indirect somatic embryogenesis was obtained on medium with 2 mg/l Kin and 0.1 mg/l BAP. Root induction was best performed on 2mg/l NAA and complete plantlets were hardened in the greenhouse before transferring to the field for further evaluation. For transformation, young leaf segments of sugarcane from the cultivar GT54-9(C9) were inoculated and co-cultivated with Agrobacterium tumefaciens strain LB4404 harboring the binary vector pISV2678 with the bar and the gus-intron genes. The obtained putative transgenic plantlets were able to grow under bialaphose containing medium. Stable integration of the bar gene into the plant genomes was tested by PCR and Southern blot hybridization. Histochemical assay and leaf painting analysis were carried out to study the expression of the gus and bar genes in transgenic plants, respectively. The results indicated that the direct organogenesis produced a higher yield of regenerated plants (22% more) within shorter time (4 weeks less). Therefore, this method is recommended for sugarcane regeneration and for further use in genetic transformation via A. tumefaciens with desired genes.

Development of a regeneration system via nodal segment culture in Veronica anagallis-aquatica L. – an amphibious medicinal plant

The present study describes an effective, reproducible and rapid regeneration protocol for in vitro shoot development of the medicinally important, semi-aquatic plant Veronica anagallis-aquatica L. through mature nodal segments on Murashige and Skoog (MS) medium. The most effective medium for shoot regeneration was MS medium fortified with 0.5 µM 6-benzyladenine (BA), on which the maximum number (43.7±1.85) of shoots per explant having a shoot length of 5.0±0.25 cm were produced. The addition of auxins along with optimal concentration (0.5 µM) of BA did not further improve the regeneration capacity of the explants; instead, it resulted in an adverse effect. Nodal segments cultured in MS liquid medium without agar provided a better and early response for efficient shoot regeneration, and simultaneous rooting as compared to solidified medium. The optimal liquid medium comprised of MS+BA (0.5 µM) on which a maximum 20.7±1.76 shoots per explant were produced and grew healthier. Both half strength MS and MS basal medium supplemented with α-naphthaleneacetic acid (NAA) responded equally for root induction. Rooted shoots were successfully transferred to field conditions with 80% survival. Histological studies revealed the de novo origin of shoots which would make this protocol suitable for transformation techniques.

A two-stage pretreatment of seedlings improves adventitious shoot regeneration in sugar beet (Beta vulgaris L.)

The effects of a two-stage pretreatment of seedlings on the subsequent shoot regeneration capacity were investigated. Pretreated seedlings were obtained by germinating seeds on three different germination media and then further culturing on six different growth media. Lamina and petiole explants of two sugar beet (Betavulgaris L.) breeding lines were then excised from the pretreated seedlings and cultured on five different shoot regeneration media. In both breeding lines, petiole explants produced significantly more shoots than lamina explants with higher frequencies of organogenic capacities; petiole explants of the lines M1195 and ELK345 produced a mean of 2.1 and 2.7 shoots per explant while their lamina explants produced 1.5 and 2.2 shoots per explant, respectively. A genotypic variation was evident as the line ELK345 was more productive for shoot development from both types of explants. In overall comparisons of different germination, growth and regeneration media, germination medium was most effective when supplemented with 0.5 mg/l 6-benzyladenine (BA) while both growth and regeneration media were most productive when contained a combination of 0.25 mg/l BA and 0.10 mg/l indole-3-butyric acid (IBA). Of all the treatments tested, the highest mean number of shoots per explant (8.3 shoots) and frequency of organogenic explants (75.6%) were obtained on regeneration medium supplemented with 0.25 mg/l BA and 0.10 mg/l IBA when petiole explants of the line ELK345 were excised from the seedlings that had been germinated on medium containing 0.5 mg/l BA followed by further growth on medium containing 0.25 mg/l BA and 0.10 mg/l IBA.

In vitro propagation of Diyarbakır watermelons and comparison of direct-seeded and transplanted watermelon

A rapid protocol using shoot tip explants for micropropagation of Diyarbakır watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] types Sürme, Beyazkış and Karakış was achieved. Shoot tips from 5-day-old in vitro germinated seedlings were cultured on shoot regeneration medium for 3 weeks. The effects of the different concentrations of benzyl adenine (BA) and carbohydrate types on shoot proliferation were examined. The results confirmed that a range of 0.5-1.0 mg/L of BA was almost equally effective in promoting the shoot length of cultures in the 3 genotypes. Sucrose, in the presence of BA, was superior to other carbohydrates for the 3 genotypes studied in terms of the number of proliferated shoots and the average shoot length obtained. The highest percentage of rooting was achieved when medium supplemented with 1.0 mg/L of indole-3-butyric acid (IBA) was used for the 3 genotypes. The highest frequency of acclimatized plantlets for the 3 genotypes was 85%, 85%, and 90%, respectively, in sterile compost, when the shoots of Beyazkış, Karakış, and Sürme were rooted in the IBA-supplemented rooting treatments. The Sürme genotype produced a significantly higher yield and mean fruit weight than the Beyazkış and Karakış genotypes in both direct-seeded and transplanted watermelons. The main soluble sugar of the experimental types was separated, identified, and quantified using high performance liquid chromatography (HPLC). Fructose was found to be the most abundant sugar and was highly detected in Beyazkış for the seeded watermelon and in Karakış for the transplanted watermelon. This demonstrates that in vitro propagation can be used to produce high quality diploid Diyarbakır watermelon for use in breeding lines.

Transgenic watermelon lines expressing the nucleocapsid gene of Watermelon silver mottle virus and the role of thiamine in reducing hyperhydricity in regenerated shoots

Viral diseases are very detrimental to watermelon production. Watermelon silver mottle virus (WSMoV) is a major limiting factor for the production of watermelon and other cucurbit fruits. There are no effective natural sources of resistance to WSMoV, making transgenic resistance an appropriate solution for attenuating virus infection. Hyperhydricity is an important problem in watermelon culture in vitro, resulting from lower multiplication rates, poor quality shoots and tissue necrosis. In this study, we report an Agrobacterium-mediated genetic transfer protocol for commercial watermelon cultivars expressing the nucleocapsid (N) gene of WSMoV and a suitable approach to overcome hyperhydricity in watermelon culture in vitro. Murashige and Skoog (MS) salts containing Schenk and Hildebrandt (SH) vitamins + 50 mg l−1 thiamine HCl could diminish the hyperhydric phenotype. The proximal halves of cotyledons from 3-day-old seedlings were cut into 1.5 × 1.5 mm segments as explants. Four days after co-cultivation, the explants were transferred to a selection medium for shoot regeneration. The putative transgenic shoots developed within 6 weeks of culture and were then transferred to stringent medium for 8 weeks to eliminate ‘escape type’ shoots. Fifty putative transgenic watermelon lines were obtained from three cultivars. PCR and Southern blot analysis confirmed that the foreign gene was incorporated into the genomic DNA of the transgenic lines.

Index Financial Time Series Based on Zigzag-Perceptually Important Points

The objective of the current study was to establish an efficient and reproducible in vitro plant regeneration protocol using cotyledonary explant for Citrullus lanatus cv. Round Dragon. To achieve optimal conditions for adventitious shoot induction, cotyledon explants of 5-day-old seedlings, 7-day-old seedlings and 9-day-old seedlings were tested for regeneration potential on Murashige and Skoog (MS) media supplemented with 2.3 mg L-1 BAP. Results showed that high frequency of in vitro adventitious shoot regeneration was induced from the proximal region of 5-day-old seedlings (93%) with 19.80±0.99 shoots per responding explant after 6 weeks. Adventitious shoots induced from 5-day-old seedlings after 6 weeks were transferred to MS shoot regeneration medium without plant growth regulator for shoot elongation for 4 weeks. The influence of various concentrations of IBA, IAA and NAA on root initiation was examined on half-strength and full-strength of MS rooting medium. The best response for root initiation was obtained from the microshoots grown in full-strength MS rooting medium compared to the half-strength MS rooting medium. Furthermore, IBA was more efficient in promoting root induction than IAA and NAA, resulting in a higher rate of root initiation (100%) at the concentration of 0.1 mg L-1 IBA. Therefore, elongated shoots were rooted in MS medium supplemented with 0.1 mg L-1 IBA for 3 weeks. Rooted plantlets were acclimatized successfully under ex vitro conditions.

Shoot regeneration and genetic transformation by Agrobacterium tumefaciens of Hydrangea macrophylla Ser. leaf discs

A reproducible procedure was developed for genetic transformation of Hydrangea macrophylla Ser. cv . Blaumeise by Agrobacterium tumefaciens following the development of an efficient regeneration system using leaf discs excised from 12 to 15 weeks old meristem-derived vitroplants. Explants were cultivated on solid B5 medium complemented with maltose 110 mM, BAP 10 μM and NAA 0.5 μM. A low light regime of 17 μmol m −2 s −1 improved regeneration frequency up to 86%. For transformation, leaf discs were inoculated and co-cultivated with two disarmed A. tumefaciens strains, EHA 101 and LBA 4404, both carrying the binary vector pFAJ3000 which contained the nptII selectable gene and the GUS reporter gene. A pre-culture period of 3 days and a short co-cultivation duration (1 day) improved the efficiency of transformation. Inoculation of only 10 min with agitation including (or not) vacuum infiltration was sufficient. If selection on kanamycin containing medium was applied after a 2 weeks culture period on shoot regeneration medium, the percentage of explants forming kanamycin-resistant shoots increased from 3.3 to 13.3%. Integration and expression of the introduced transgene were confirmed by histochemical GUS assay, PCR and Southern blot analysis. Flowering of transgenic plants in glasshouse occurred 10 months after acclimatization.

Doubled haploid callus lines of Valencia sweet orange recovered from anther culture

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial plants using conventional breeding techniques is currently a challenge because of a long juvenile period, high heterozygosity and the substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In this study, we report the regeneration of doubled haploid lines of Valencia sweet orange cv. Rohde Red (Citrus sinensis [L.] Osbeck) via anther culture. Anthers at the uninucleate stage were induced and two embryogenic calli were obtained that further regenerated to embryoids (2/400). Plantlets were obtained after transferring the embryoids to a shoot regeneration medium, but were short-lived. Ploidy analysis via both flow cytometry and chromosome counting verified that these two lines were diploids. Additionally, 43 simple sequence repeat (SSR) markers which showed to be heterozygous in the Valencia sweet orange donor line confirmed homozygosity and doubled haploids in the anther-derived lines. Furthermore, analysis of the doubled haploids via cleaved amplified polymorphic sequence (CAPS) markers and target region sequencing confirmed the allelic state of two genes (LCYE and LCYB) involved in the carotenoid biosynthesis of sweet oranges.

A micropropagation protocol for Cassia angustifolia Vahl. from root explants

The aim of this study was to develop a new micropropagation system for Cassia angustifolia Vahl., an important medicinal legume using root explant as starting material. Root explants taken from 30-day-old aseptic seedlings were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators: 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Organogenic nodular calli obtained on MS + TDZ (1.0 μM) were transferred to shoot regeneration medium supplemented with different cytokinins (BA, Kn or TDZ) either alone or in combination with auxin:indole-3-acetic acid or α-naphthalene acetic acid. Maximum shoot regeneration frequency (90%) was obtained on MS + BA (2.5 μM) + NAA (0.6 μM) wherein a maximum of 42.76 ± 1.47 shoot buds per explant were induced with a maximum conversion rate of 35.63 ± 0.75 shoots per explant and average shoot length of 5.43 ± 0.20 cm. Elongated microshoots were successfully rooted under ex vitro conditions by pulse treatment in 200 μM of indole-3-butyric acid for half an hour. Microshoots were rooted, acclimatized and hardened off simultaneously in sterilized soilrite inside the growth room and then established in pots containing sterilized soil and manure (1:1) and grown under greenhouse condition with 90% survival rate. The histological sections at different developmental stages of shoot buds revealed the organization of nodular meristematic zone leading to the orientation and differentiation of shoot buds in large number and thereafter conversion into healthy shoots.

Enhanced resistance against bacterial wilt in transgenic tomato (Lycopersicon esculentum) lines expressing the Xa21 gene

To enhance bacterial wilt resistance in tomato plants and simplify the protocol of Agrobacterium tum- efaciens mediated gene transfer, parameters affecting transformation efficiency in tomato have been optimized. A. tumefaciens strain EHA101, harboring a recombinant binary expression vector pTCL5 containing the Xa21 gene under the control of the CaMV 35S promoter was used for transformation. Five cultivars of tomato (Rio Grande, Roma, Pusa Ruby Pant Bahr and Avinash) were tested for transformation. Transformation efficiency was highly dependent on preculture of the explants with acetosyrin- gone, acetosyringone in co-cultivation media, shoot regeneration medium and pre-selection after co-cultivation without selective agent. One week of pre-selection fol- lowing selection along with 400 lM acetosyringone resulted in 92.3% transient GUS expression efficiency in Rio Grande followed by 90.3% in Avinash. The presence and integration of the Xa21 gene in putative transgenic plants was confirmed by polymerase chain reaction (PCR) and Southern blot analyses with 4.5-42.12% PCR-positive shoots were obtained for Xa21 and hygromycin genes, respectively. Transgenic plants of the all lines showed resistance to bacterial wilt. T1 plants (resulting from self-

The effect of plant growth regulators, explants and cultivars on spinach (Spinacia oleracea L.) tissue culture.

Spinach (Spinacia oleracea L.) is an important vegetable crop of which dioecy in nature has made cultivar improvement difficult using traditional breeding methods; therefore, production of high amount of disease free spinach is critical. To achieve the best explants and media for spinach tissue culture, the effects of two different plant growth regulators, two explants and cultivars on adventitious shoot regeneration were tested. The Analysis of Variance (ANOVA) showed that the effects of plant growth regulators on spinach tissue culture were significant; moreover, the effects of explants were not significant except on the regeneration phase. The best medium for callous induction was MS media containing 1.5 mgl-1 IAA + 2.5 mgl-1 GA3. The best medium for shoot regeneration was MS media contained 0.5 mgl-1 NAA + 2 mgl-1 GA3. The best rooting medium was MS medium containing 0.5 mgl-1 IBA. Results presented inhibitory effect of GA3 for callus and root formation; whereas, promote shoot development.