Protocol For Organogenesis Research Articles

Optimized Direct Organogenesis from Shoot Tip Explants of Date Palm.

In vitro propagation is an available alternative to produce uniform and good-quality planting material to establish large-scale date palm cultivation in a short time. This study was carried out to achieve organogenesis and multiplication directly from shoot tips without callus formation, thus avoiding any possibility of undesirable genetic variability among the regenerated plants. The shoot tips explants are cultured on Murashige and Skoog (MS) medium supplemented with 1mg/L naphthaleneacetic acid (NAA), 1mg/L naphthoxyacetic acid (NOA), 2.5mg/L benzyladenine (BA), and 2.5mg/L isopentenyladenine (2iP). Numerous adventitious buds appeared from the shoot tip explants in darkness after six subcultures at 4-week intervals. Vegetative buds pass through three stages: initiation bud formation, vegetative bud differentiation, and shoot bud proliferation. Shoots are transferred onto medium containing low concentrations of growth regulators for shoot multiplication. The organogenesis protocol described herein consists of six steps: initiation of meristematic buds, multiplication, elongation, rooting, pre-acclimatization, and finally plant acclimatization.

Organogenesis and evaluation of genetic homogeneity through SCoT and ISSR markers in Helicteres isora L., a medicinally important tree

The present study develops the highly reliable and reproducible protocol for indirect organogenesis of Helicteres isora L., a medicinally important multipurpose plant of Sterculiaceae family. Murashige and Skoog (MS) medium supplemented with different plant growth regulators induced callus with different type and texture. The combination of 10.74μM naphthalene acetic acid (NAA) and 5.71μM indole-3-acetic acid (IAA) fortified with MS medium resulted best response (100%) in callus induction with fresh weight from leaf (3.75±0.11g) and internodal (3.30±0.14g) explants. Green compact nodular callus (GCNC) was transferred to shoot regeneration medium for shoot regeneration. The multiplication rate of adventitious shoots was influenced by various factors like explant type, media composition, plant growth regulator combinations and concentrations. MS medium supplemented with 2.69μM NAA, 0.57μM IAA and 4.92μMN6-(2-isopentenyl) adenine (2ip) combination resulted in 80.95±4.76 percentage of response with 8.43±0.43 number of shoots per piece of callus derived from leaf explant. This combination was expressed 76.19±4.76 percentage of response with 7.28±0.28 number of shoots per internode-derived callus respectively. The addition of 50 mg l−1 glutamine in 2.69μM NAA, 0.57μM IAA and 4.92μM 2iP combination enhanced the shooting frequency with 12.14±0.83 and 9.14±0.51 shoots from leaf and internodal explant-derived callus. Elongated shoots [4.92μM 2iP and 1.44μM of gibberellic acid (GA3)] were achieved rooting in half-strength MS medium fortified with 4.90μM indole-3-butyric acid (IBA) with 582.84μM activated charcoal. The genetic fidelity between mother plant and in vitro plants was assessed through SCoT and ISSR marker systems. Both these analysis revealed that all the samples were found monomorphic in nature. This suggests that the current study standardised the true-to-type culturing protocol and authenticated the in vitro raised plantlets are remain free from somaclonal variations.

Micropropagation of Eucalyptus grandis × E. urophylla AEC 224 clone

Genetic transformation systems require protocols that allow regenerating transgenic plants from transformed tissues. This study aimed to establish a protocol for indirect organogenesis in leaf explants of a Eucalyptus grandis × E. urophylla AEC 224 clone. During callogenesis stage, several concentrations of NAA and then NAA or 2,4-D combined with TDZ were tested in JADS culture medium for 30 days, followed by subculture of the explants in the regeneration medium, containing 5.0 µM BA and 0.5 µM NAA for another 30 days. In these media, the explant oxidation rate was high (95 %). Thus, in order to reduce oxidation, different culture media were compared: WPM, MS, JADS and modified QL, followed by explant transfer onto regeneration medium. The highest percentage of regeneration and the lowest oxidation rate were achieved on WPM medium. Then, NAA and 2,4-D were tested in combination with TDZ and also TDZ and BA combined with NAA in WPM medium. The most efficient culture media in terms of shoot regeneration were WPM supplemented with 0.25 µM TDZ and 0.1 µM NAA during 30 days for callus induction and then with 5.0 µM BA and 0.5 µM NAA for another 30 days. This protocol yielded a regeneration rate of 43 %, with a low oxidation of tissues. A rooting experiment was conducted using half strength MS medium and comparing three concentrations of IBA (2.46, 4.90 and 7.35 µM). The highest rooting percentage (35 %) was obtained on medium containing 2.46 µM IBA. Once the shoots were rooted, acclimatization in a greenhouse was not challenging and plant survival reached 100 %.

Epicotyl sections as targets for plant regeneration and transient transformation of common bean using Agrobacterium tumefaciens

Common bean is recalcitrant to genetic transformation, due to limited regeneration capacity and low DNA transfer rates. The effect of different parameters on T-DNA transfer from Agrobacterium tumefaciens, was studied by measuring transient expression of the β-glucuronidase gene in Phaseolus vulgaris L. cv. CIAP7247F. Epicotyl containing seedling explants were inoculated with Agrobacterium EHA101 and C58C1RifR(pMP90) strains harboring the binary vector pTJK136 with GusA gene on the T-DNA. Parameters studied were temperature and light regime during co-cultivation, explant injury, and the acetosyringone concentration for vir gene induction. The co-cultivation temperature and photoperiod had a significant effect on Agrobacterium DNA transfer. In addition, explant injury and supplementation of the co-cultivation medium with acetosyringone increased the GUS activity. Optimal T-DNA transfer was obtained under the following conditions: co-cultivation at 25°C in darkness, injuring the explants with carborundum, and supplementation of the co-cultivation medium with 200 μM acetosyringone. This T-DNA delivery system was combined with a direct organogenesis protocol using epicotyl explants and fertile regenerants were recovered from tissue transformed with Agrobacterium. However, no transmission of transgenes to progeny could be observed, suggesting that the obtained plants were chimeras.

English

Melia dubia Cav. (Meliaceae) is a multipurpose tree of tropical and subtropical regions mainly cultivated for its medicinal and industrial importance. Due to its versatile properties, it has been depleted in its natural environment. Moreover due to sluggish and poor seed germination, there is a threat of its gene pool exclusion from the natural habitat. The alternative method for conservation and efficient mass propagation is thus need of the hour. As per the extensive literature survey there is no report on efficient protocol for mass propagation of M. dubia through callus organogenesis. Therefore, the present work was aimed to develop in vitro organogenesis protocol for rapid and large scale production of planting material. From our results, maximum callus percentage, callus weight and fragile callus was observed on 1.0 mg/l benzylaminopurine (BAP) in combination with 0.5 mg/l naphthalene acetic acid (NAA). The callus differentiation was achieved at different concentrations of BAP and indole acetic acid (IAA). Multiple Shoot number per callus propagule 5.30 was observed on 0.5 mg/l BAP and 1 mg/l IAA concentration. The maximum rooting percentage (78.5%), root number per explant (4.33) and root length per explant (4.41 cm) was observed at 0.5 mg/l indol butyric acid (IBA) after 30 days of inoculation. Further the total flavonoid content, phenolic content and antioxidant properties of leaves of in-vitro regenerated plants where studied. Total flavonoids and phenolic content in leaves of in vitro Melia dubia was 0.56 ± 0.8 mg quercitin equivalent (QE) and 2.97 ± 0.17 mg gallic acid equivalent (GAE) respectively. The antioxidant property was further assed through measurement of DPPH radical scavenging activity. The in-vitro regeneration protocol can be exploited for commercial cultivation and fulfilling the growing demand for fresh explant material through mass propagation of M. dubia an economically important plant species. Key words:  Melia dubia, antioxidant, indole-3-butyric acid, flavonoids and phenolics.

Indirect in vitro organogenesis of Hancornia speciosa Gomes

Hancornia speciosa Gomes is a fruit species belonging to the Apocynaceae family and holds social, cultural and economic potential mostly due to its use in composition of many food industry products and the consumption its fruits in natura . Several aspects regarding their propagation need further studies, since the species is undergoing a continuous process of domestication. The objective was to obtain an in vitro protocol for indirect organogenesis and rooting with subsequent acclimatization of H. speciosa plants. To obtain indirect organogenesis, internodal segments were inoculated in WPM culture medium gelled with 7 g L -1 agar, added with 30 g L -1 sucrose, 0.4 g L -1 PVP, and supplied with different concentrations of 2,4-D (0.0; 2.46; 7.38; 12.30; and 17.22 µM), BAP (0.0; 4.92; 9.84; 14.76; and 19.68 µM), and TDZ (0.0; 4.92; 9.84; 14.76; and 19.68 µM). For the in vitro rooting, shootings with approximately 6.0 cm diameter length kept for 15 days in WPM medium with no plant growth regulator and, afterwards, subjected to treatments with different auxins (Control; 9.84 µM IBA; 9.84 µM NAA; and 9.84 µM IAA) as well as the combination among them, in order to verify their effects on percentage of rooting (%), number of roots, and average length of the largest root (cm). The formation of calluses was observed in all explants subjected to the concentrations of the regulators tested. The highest shooting regeneration occurred with 7.38 µM 2,4-D (73%). The highest percentage of shoot rooting (80%) and roots with the largest length (1.3 cm) were found in the culture medium with the combination of 4.92 µM NAA and 4.92 µM IBA. The in vitro regeneration of H. speciosa is feasible. The acclimatization of rooted shoots in Trospstrato® was accomplished with successful and 100% survival of plant material was observed during this stage.

Indirect organogenesis and assessment of somaclonal variation in plantlets of Vanilla planifolia Jacks

The low genetic variability of vanilla (Vanilla planifolia Jacks.) makes it susceptible to pests and diseases, which leads to a decrease in production. The genetic variability can be broadened by using in vitro plant tissue culture techniques. The use of indirect morphogenic routes allows increasing the percentage of somaclonal variation in regenerated plantlets. The objective of this research was to establish a protocol for indirect organogenesis in V. planifolia aimed at broadening the genetic base of the crop. A friable callus was induced from immature seeds grown in the dark, using MS medium supplemented with 2.27 μM thidiazuron (TDZ). Subsequently, 6.8 shoots per callus were regenerated in MS medium supplemented with 8.88 μM benzyladenine (BA). One hundred per cent rooting of regenerated shoots was achieved when MS medium was used with no plant growth regulators. Last, rooted plantlets were acclimatized in a greenhouse. A 91 % survival rate was observed. Molecular analyses on regenerated plantlets revealed the existence of a 71.66 % genetic polymorphism. Furthermore, morphological variation was confirmed by the presence of variegated individuals in regenerated plantlets. The proposed protocol can be useful in subsequent vanilla genetic improvement works.

Ayçiçeği (Helianthus annuus L.)’nin in vitro Organogenezi Üzerine Etilen İnhibitörleri Olarak AgNO3 ve CoCl2’nin Uyarıcı Etkisi

To achieve an effective and reliable shoot organogenesis protocol in Helianthus annuus L., effect of ethylene inhibitors including AgNO3 and CoCl2 were investigated. Cotyledonary explants of two cultivars including CMS19 and Progress were cultured in MS medium with 2.0 mg L-1 BAP and 1.0 mg L-1 IAA supplemented with different concentrations of AgNO3 (0, 1.5 and 3 mg L-1) and CoCl2 (3 and 6 mg L-1). Statistical analysis of data showed a significant difference among cultivars and ethylene inhibitors concentrations for organogenesis parameters including shoot regeneration and number of shoots per explant. Addition of ethylene inhibitors to culture medium improved regeneration frequency and number of shoots per explant. Shoot generation was improved with increasing concentrations of CoCl2 but in the case of AgNO3, increasing the concentration gave adverse results. Treatment of explants with AgNO3 in low concentration (1.5 mg L-1) improved organogenesis in Helianthus annuus. The highest shoot regeneration was obtained in media supplemented with 6 mg L-1 CoCl2 (R/E= 63% and S/RE= 3) and 1.5 mg L-1 AgNO3 (R/E= 73% and S/RE= 2).This study suggests that ethylene inhibitors, particularly CoCl2, could be used to improve the efficiency of in vitro shoot regeneration and plant transformation protocols of sunflower. Key words: Cotyledon, Silver nitrate

Use of induced pluripotent stem-cell technology to understand photoreceptor cytoskeletal dynamics in retinitis pigmentosa

BackgroundRetinitis pigmentosa, which affects one in 3000 people, causes blindness and has no treatment. Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause 20% of all cases. Recent work suggests that RPGR, localised to the photoreceptor connecting cilium, regulates rhodopsin transport to the outer segment through its effect on the turnover of actin. We set out to establish a novel model for RPGR disease to test the hypothesis that RPGR mutations lead to retinal degeneration due to a dysregulation of the actin cytoskeleton. MethodsPatients with RPGR mutations and their unaffected relatives were recruited and skin biopsy samples taken. Fibroblast lines were established and reprogrammed to generate induced pluripotent stem cell (iPSC) lines. A three-dimensional organogenesis protocol was optimised whereby embryoid bodies were formed and patterned towards an eye field fate in a 100-day retinal differentiation protocol, allowing three-dimensional optic cups to form. RPGR-mutated cultures were compared with their healthy controls. FindingsMutant and wild-type iPSC lines were generated and characterised. Differentiation of all lines resulted in the generation of optic cups in a self-organising manner after 100 days in culture. These cultures contained mature photoreceptors, as evidenced by morphology and both RNA and protein expression. Photoreceptor cultures from RGPR-mutated iPSCs had increased actin polymerisation compared with controls (mean confocal pixel intensity count 59·02 [SD 16·24] vs 23·70 [8·20], p=0·0081). This finding was confirmed by assessment of F-actin with western blot. Pathways regulating actin turnover were explored; western blot analysis showed a reduction in both Src and ERK phosphorylation in RGPR-mutated photoreceptor cultures. An unbiased protein array confirmed this reduction in ERK and Src activation. Several other pathways were also shown to be dysregulated in the RGPR-mutated photoreceptor cultures. InterpretationThis study supports the hypothesis that RPGR mutations lead to actin dysregulation. We have identified several pathways that are interrupted in RPGR-mutant photoreceptor cultures and could be contributing to disease. This study is the first use, to our knowledge, of human iPSCs with retinitis pigmentosa-causing mutations to look at pathophysiology of disease. FundingWellcome Trust.

Abscisic acid promotes shoot regeneration in Arabidopsis zygotic embryo explants

An efficient shoot organogenesis protocol for Arabidopsis zygotic embryo explants of Landsberg erecta ecotype was established. This de novo shoot organogenesis protocol has three different steps, i.e., induction of callus in an auxin-rich callus induction medium, the formation of green-organogenic callus in the shoot induction medium (SIM), and the final morphological differentiation of shoot in the hormone-free shoot development medium (SDM). Abscisic acid (ABA), auxin, and cytokinin (CK) were used in the SIM. Individual plant growth regulators as well as their combination were studied to understand their importance in the shoot induction treatment. We found that a combination of ABA + CK and ABA + CK + auxin induced higher shoot organogenic ability in the callus than ABA, CK, and auxin alone. Optimum ABA concentration on shoot organogenesis was determined to be 10−5 M. Morphological characterization of callus induction and shoot organogenesis events indicated that calli were derived from the cotyledons of zygotic embryo explants and the formation of green organogenic calli was specific to the exogenous inclusion of ABA + CK in the SIM. During the time of shoot development, the green organogenic callus became darker green due to the formation of anthocyanins. Shoot organogenic calli in the SIM and the SDM were easily identified by the green-colored calli and anthocyanin pigments, respectively. Furthermore, we demonstrated the significance of exogenous and endogenous ABA in shoot organogenesis by fluridone treatments. The inclusion of ABA in SIM has a significant effect on shoot formation.

Indirect Organogenesis and Histological Analysis of Garcinia mangostana L.

Abstract: The improvement of Garcinia mangostana L. through biotechnology approach needed an efficient plant regeneration system. The objective of this research was development of indirect organogenesis protocol for mangosteen such as effect of plant growth regulator for induction of nodular calli, plant regeneration and histological analysis of nodular calli and shoot bud. The treatments for induction nodular calli were concentration of BAP 2.2 and 4.4 uM while concentration of TDZ (thidiazuron) 1.14, 2.27 and 4.54 p.M. Combination of BAP and TDZ concentration used as treatments. The medium of shoot proliferation was Woody Plant Medium (WPM) with concentration of BAP (0.0; 1.1; 2.2; 3.3; 4.4) uM as treatments. All experiments were arranged completely randomized design and analyzed data using F-test and the means among each treatment was separately by Duncan's Multiple Range Test (DMRT). A result showed that combination of 2.22 and 2.27 itM TDZ on MS medium produce nodular calli basal medium WPM with 2.2 itM BAP concentration indicated the highest percentage of nodular callus formed shoot was 34.7%, average of numbers shoot per nodular calli was 7.8 shoots, average of time formed shoot was 94.5 days and number of shoot length 1-5 mm was 11.06, shoot length 6-10 mm was 2.61 and shoot length >10 mm was 0.61. The highly efficient protocol of indirect organogenesis suggested in this study can be used to mutation breeding methods and propagation of Garcinia mangostana L.

Organogênese in vitro de batata (Solanum tuberosum L.) cultivar Atlantic visando transformação genética

The obtaining of commercial cultivars of potato with resistance to the phytopatogen is a very promising alternative that the genetic engineering can provide, through the introduction of exogenous genes for the genetic transformation. In order to establish an efficient system of transformation, it is crucial first to optimize in vitro organogenesis protocol. This work aimed to establish an efficient protocol for the genetic transformation of potato cv. Atlantic. The efficiency of the in vitro organogenesis is influenced by the explant type and by the kind and concentrations of growth regulators used. Shoot segments of potato cv. Atlantic present better organogenesis capacity than leaf explants. It is recommended to use the woody plant medium (WPM) supplied with 1,0 mg L-1 of naphthalene acetic acid (NAA) + 5,0 mg L-1 of zeatin riboside (ZEA), to obtain shoots from shoot segments, and the carbenicilin (Cb) added to this medium increased this formation. For the process to enlarge shoots, this same antibiotic when added to the WPM medium in growing concentrations (100; 250; 500 mg L-1) promoted a decrease in the height of plants, number of shoots and length of roots. After the co-cultivation of shoots segments with Agrobacterium tumefaciens these concentrations of Cb are not efficient in the elimination of the bacteria, which commits the organogenesis. However, it is possible to establish a protocol of in vitro organogenesis of potato cv. Atlantic starting from shoots segments not co-cultivated. These results will be useful for futures experiments of genetic transformation with this and another commercials cultivars of potato.

Organogênese in vitro de batata (Solanum tuberosum L.) cultivar Atlantic visando transformação genética

The obtaining of commercial cultivars of potato with resistance to the phytopatogen is a very promising alternative that the genetic engineering can provide, through the introduction of exogenous genes for the genetic transformation. In order to establish an efficient system of transformation, it is crucial first to optimize in vitro organogenesis protocol. This work aimed to establish an efficient protocol for the genetic transformation of potato cv. Atlantic. The efficiency of the in vitro organogenesis is influenced by the explant type and by the kind and concentrations of growth regulators used. Shoot segments of potato cv. Atlantic present better organogenesis capacity than leaf explants. It is recommended to use the woody plant medium (WPM) supplied with 1,0 mg L-1 of naphthalene acetic acid (NAA) + 5,0 mg L-1 of zeatin riboside (ZEA), to obtain shoots from shoot segments, and the carbenicilin (Cb) added to this medium increased this formation. For the process to enlarge shoots, this same antibiotic when added to the WPM medium in growing concentrations (100; 250; 500 mg L-1) promoted a decrease in the height of plants, number of shoots and length of roots. After the co-cultivation of shoots segments with Agrobacterium tumefaciens these concentrations of Cb are not efficient in the elimination of the bacteria, which commits the organogenesis. However, it is possible to establish a protocol of in vitro organogenesis of potato cv. Atlantic starting from shoots segments not co-cultivated. These results will be useful for futures experiments of genetic transformation with this and another commercials cultivars of potato.

A more improved protocol for in vitro shoot organogenesis in daylily (Hemerocallis sp.)

Daylily is a crop of landscape, nutritional, and medicinal importance. Despite relative progress, daylily is one of the least explored crops in tissue culture, partly, because of its low responsiveness for in vitro organogenesis. This study reports a more efficient one-step protocol for shoot organogenesis. Leaf, flower, flower bud, stem, and root tissues were investigated for shoot organogenesis after different growth regulator treatments on Murashige and Skoog medium. Three out of four of the daylily formed shoots. The variety 'Summer Echoes' was the best of all the other varieties with the greatest average shoots per explant (118) and shooting explant rate 87% when 6-benzyl-aminopurine (BA) and kinetin were used singly. This protocol is the most improved, over the current ones, for shoot organogenesis in daylily. The plant grew normally in the greenhouse. Keywords : Daylily shoots organogenesis, plant micropropagation, daylily tissue culture, in vitro plant regeneration African Journal of Biotechnology Vol. 12(8), pp. 820-825

In vitro organogenesis from leaf and transverse thin cell layer derived callus cultures of Talinum triangulare (Jacq.) Willd.

Talinum triangulare is an important medicinal herb used traditionally in the treatment of various diseases. The present study was intended to develop a rapid and efficient protocol for indirect organogenesis from leaf discs and transverse thin cell layer (tTCL) of internodal explants of T. triangulare. Best callusing response (100 %) was observed with tTCL explants on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyl amino purine and 5.37 μM α-naphthalene acetic acid (NAA). High frequency shoot regeneration (96.67 %) was obtained from tTCL derived calli on MS medium supplemented with a combination of 0.45 μM thidiazuron and 0.27 μM NAA, by producing 9.20 ± 0.35 shoots with a shoot length of 2.74 ± 0.03 cm. In vitro rooting of the microshoots was recorded on half-strength MS medium containing 2.46 μM indole-3-butyric acid by eliciting 15.20 ± 0.27 roots with a length of 4.25 ± 0.11 cm. The rooted shoots were acclimatized on garden soil, sand and coco pith (1:1:3 v/v) planting substrate. The plantlets were successfully established under field conditions with 100 % survival rate. The hardened plants exhibited homogeneity and no observable morphological variations were detected among the regenerants and the mother plants of T. triangulare.

In Vitro Organogenesis from Cotyledon Derived Callus Cultures of Punica granatum L. cv. Kandhari Kabuli

An efficient protocol for shoot organogenesis and plant regeneration has been developed for Punica granatum L. cv. Kandhari Kabuli using cotyledon derived callus. Calli were induced from cotyledon explants procured from in vitro germinated seedlings on MS medium supplemented with various concentrations and combinations of plant growth regulators. Optimal callus formation (93.71 %) from cotyledon explants was obtained on MS medium supplemented with 4.0 mg/l BAP and 3.0 mg/l NAA. The highest shoot induction response (72.34 %) was recorded on MS medium containing 2.0 mg/l BAP and 2.0 mg/l NAA leading to maximum average no. of shoots per callus clump (7.16) and shoot length (1.98 cm). The highest percentage of root regeneration (72.50 %) was observed on MS medium supplemented with 0.10 mg/l NAA. The in vitro raised plantlets were successfully acclimatized and grew well in the greenhouse. All the regenerated plantlets appeared normal with respect to morphology and growth characteristics with 80 % survival rate.

Development of somaclones in sugarcane genotype BF-162 and assessment of variability by random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers in selected red rot resistant somaclones

Worldwide, sugarcane ( Saccharum officinarum L) is the major source of commercial sugar along with many other value added products. In Pakistan, during the year 2008 to 2009, there was a production of 50.05 million tonnes. Sugarcane genotype BF-162 was released for general use in the Punjab province during 1990, and it became susceptible to red rot. As environmental conditions are not conducive for flowering, so the red rot rectification was tried through somaclonal variation. Protocol for callogenesis and organogenesis was standardized. Leaf when used as explant source and 2,4-dichlorophenoxyacetic acid (2,4-D) as auxin at 3mg/l performed better in callogenesis. It was observed that lower doses of Kinetin regenerated more numbers of shoots, while indole-3-butyric acid (IBA) developed more numbers of roots. Red rot resistance somaclones were isolated and assessed for the presence of variability through random amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR) markers. Polymorphism captured by RAPD was 33.73% and by SSR was 64%. Polymorphic information content (PIC) value ranged between 0.02 and 0.45 for RAPD and 0.12 and 0.49 for SSR. Cluster and sub cluster formation further verified the presence of variability in the red rot resistant somaclones with respect to the parent. Key words : Sugarcane, callogenesis, organogenesis, somaclone, polymorphism, cluster.

Rapid in vitro plant regeneration from leaf explants of Launaea sarmentosa (Willd.) Sch. Bip. ex Kuntze

An efficient protocol for organogenesis through leaves has been established for Launaea sarmentosa (Willd.) Sch. Bip. ex Kuntze, a highly valuable medicinal plant. The leaf explants produced microshoots on MS basal medium when fortified with cytokinins and auxins. A combination of 6-benzylaminopurine (BAP) at 0.5mg/l and naphthaleneacetic acid (NAA) at 0.2mg/l resulted in the induction of high frequency microshoots in 30 days. The microshoots were successfully subcultured for shoot elongation and eventually for rooting on MS medium supplemented with indole-3-butyric acid (IBA) at 0.5mg/l. The regenerated plantlets were hardened under greenhouse conditions and transferred to garden, resulting in a 90% survival rate.

Development of Eucalyptus tissue culture conditions for improved in vitro plant health and transformability

Background Despite its importance as a widely-planted crop tree, eucalypt species and hybrids are relatively difficult to micropropagate, culture and genetically transform in vitro. Compared to other plant species, few non-commercial laboratories are proficient at Eucalyptus tissue culture and transformation. We have undertaken to establish and transform several eucalypt clones in the laboratory. Our main aims include the identification of clones amenable to culturing and transformation, and the development of robust and transferable micropropagation, organogenesis and transformation protocols to enable routine production of transgenic eucalypts for public sector research. Efficient transformation protocols are essential to take full value of the eucalypt genome for functional genomics, ecophysiology, and biotechnology.

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.