Shoot Induction Medium Research Articles

Thin Cell Layer (TCL) Culture System for Herbal Biomass Production and Genetic Transformation of <i>Bacopa monnieri</i> L. Wettst.

Bacopa monnieri (L.) Wettst. (Scrophulariaceae) is a highly sought after medicinal plant with therapeutic properties as cognition enhancer as well as for other brain and body functions. Research was conducted to optimize a thin cell layer explant based micropropagation system to assist mass propagation. Thin cell layers (TCL) derived from leaf and internode segments were used as explants. Murashige and Skoog medium was used to formulate shoot induction, elongation, and rooting media. Shoot induction media were prepared by supplementing three concentrations (0.1, 1.0, and 10.0 μM) of four cytokinins 6-benzylaminopurine, 2-isopentenyl-adenine, 6-3-Hydroxybenzylaminopurine, and thidiazuron to study adventitious shoot bud induction response. An optimum shoot bud induction response was observed on MS medium supplemented with 10.0 μM 6-benzylaminopurine for both leaf and stem transverse thin cell layer (tTCL) explants. The average number of shoot buds from leaf tTCL explants was 59, whereas, on an average, 33 shoot buds were regenerated from internode tTCL explants. Elongation of adventitious shoot buds was achieved best in a liquid medium using Liquid Lab Rocker® system. Elongated shoots recorded 100% rooting in MS medium supplemented with 5 μM indole butyric acid. Bacopa micropropagation employing tTCL explants for initial shoot bud induction and using LLR® boxes in subsequent elongation step can achieve cost effective way to regenerate high volume of plantlets and biomass required for herbal industry. Leaf and stem tTCL explants both were suitable for Agrobacterium tumefaciens (EHA105) mediated genetic transformation. Successful transformation was scored within three days of co-cultivation with Agrobacterium suspension on the basis of Enhanced Green Fluorescent Protein (EGFP) expression as an early and non-destructible screening device. Transformation frequencies of 83% and 76% were accomplished for leaf and stem tTCL explants, respectively. Greenhouse grown Bacopa plants were analyzed as fresh and dry methanolic extracts for total polyphenol content (811.93 ± 7.98 and 814 ± 17.64 GAE mg g-1) and the Trolox Equivalent Antioxidant Capacity values were 1918.25 ± 173.12 and 3163.14 ± 403.25 μmol/g, respectively.

Refined glufosinate selection and its extent of exposure for improving the Agrobacterium-mediated transformation in Indian soybean (Glycine max) genotype JS-335.

Soybean like many other crops, in this genomic era, has well-established genomic database which provides a wide range of opportunities for improvement through genetic manipulation. But the growing demand for soybean transgenics with increased production and improved quality has been handicapped due to inefficient transformation strategies and hence an efficient, stable and reliable transformation system is of prime requisite. In the present study, Agrobacterium-mediated transformation was standardized by refining the glufosinate selection system in terms of dosage (0-6 mg l-1) and degree of exposure. The cotyledonary node explants (with and without wounding) initially cultured on a non-selective shoot induction medium for 10 days before transferring them to the selective SIM with an optimized concentration of 5.0 mg l-1 ammonium glufosinate, showed least selection escape frequency. Wounded cotyledonary node explants infected with Agrobacterium tumefaciens harboring pBIN-bar construct, showed an improved regeneration efficiency of 55.10% and transformation efficiency of 12.6% using Southern blotting in T1 plants. Southern analysis of T1 plants confirmed the integration of bar gene into the genomic DNA and the bar positive T1 plants segregated in 3 : 1 ratio. This is the first report, to our knowledge, of a high transformation efficiency using Agrobacterium-mediated cot node-glufosinate system in an Indian soybean genotype.

Effect of plant growth regulators on in vitro plant regeneration of sandalwood (Santalum album L.) via organogenesis

Santalum album L. is an economically important tropical tree species. Sandalwood trees are the source of highly priced and fragrant heartwood, the ‘East Indian Sandalwood’ that on steam distillation yields on an average 5–7 % oil of high perfumery value. Owing to extensive logging, changes in land-use patterns and poor natural regeneration, the natural sandal populations are rapidly dwindling. An efficient protocol of plant regeneration from node explant has been developed for S. album. Optimal callus was developed from nodal segments on woody plant medium (WPM) supplemented with 0.6 mg 1−1 TDZ and 1.5 mg 1−1 2,4-D. Fresh weight (132.3 mg) and dry weight (41.0 mg) of node derived callus were highest in medium supplemented with 0.6 mg l−1 TDZ. Shoot bud initiation was achieved from the surface of callus when transferred on shoot induction medium supplemented with BA and NAA in combination. Highest number of shoot buds (16.0) per callus was observed in medium containing 2.5 mg l−1BA + 0.4 mg 1−1 NAA. Shoot proliferation (number of shoots/callus) (21.5) and shoot length (4.7) was highest in media containing 3.0 mg 1−1 Kn. Plantlets were rooted on WPM medium with different concentrations of indole-3-butyric acid (IBA). Highest rooting percentage (82.37) and survival was achieved on WPM media with 1.5 mg 1−1 IBA. The in vitro raised plantlets with properly developed shoot and roots were acclimatized successfully and grew well in the greenhouse. All the regenerated plants appeared normal with respect to morphology and growth characteristics.

IDENTIFICATION OF BULBING HORMONE GENES IN ONION (Allium cepa)

<p>Initiation of onion (Allium cepa L.) bulb formation seems to require “bulbing hormone” which has been considered to be produced in leaf blades in response to the stimulus of long day conditions. But “bulbing hormone” is not yet identified. Previous study revealed a protein called Flowering locusT (FT) as flowering hormone, florigen. FT act for flowering by change on the day length on the higher plants. Objective of this study is identify”bulbing hormone” in onion plants. Method used in this study are cloning gene and gene expression analysis of the FT in onion plants.Full length of cDNA was cloned by the degenerate PCR and 5’­and 3’-RACE method.As a result, six kinds of full length cDNA clones for FThomologs in onion plants were obtained.These genes were named AcFT1 to6. By expression analysis of these genes, AcFT4, 5 and 6,expression increased as it got closer to a condition in long days in association with the bulbing of onion. Furthermore, in order to investigate the functions of these genes, we optimize transformation methods for onion plants. Medium containing 2,4-Dand kinetin showed high efficient plant regeneration from seed-derived callus of onion. Medium containing 2,4-D and kinetin as plant growth regulators is effective for induction of shoot-inducible callus, and advance shoots were developed from the callus on the shoot induction medium which contained thidiazuron, benzyl adenine or trans-zeatin as cytokinins. In conclusion, bulbing hormone in onion plants were possibly gene AcFT4, 5,6.</p><p><br /><strong>Keywords</strong> : Allium cepa L, bulbing hormone gene, Flowering locusT</p>

Sunflower (Helianthus annuus L.) organogenesis from primary leaves of young seedlings preconditioned by cytokinin

Leaf tissue is widely used for regeneration of many different plants, but an efficient regeneration method using leaf tissue of sunflower is not yet available. In this report, adventitious shoots were obtained from primary leaves of 7-day-old sunflower seedlings. If seeds were germinated on a medium without cytokinin, only 25 % of the leaf explants produced shoots after 3 weeks of culture on a shoot induction medium (SIM) containing 1.5 mg L−1 benzylaminopurine (BA), giving an average of three shoot primordia per leaf. Shoot induction was enhanced up to sixfold when seeds were germinated on media containing BA or 6-(γ, γ-dimethylallylamino)purine at 5 mg L−1 and higher, or kinetin at 15 mg L−1 and above, with an average of 12–19 shoot primordia per leaf. Shoots further developed and elongated following transfer to a medium containing 0.1 mg L−1 gibberellic acid. The highest number of developed shoots was obtained from leaves preconditioned with 5 mg L−1 BA, with 1.9 developed shoots per explant. Histological analysis of leaves from seedlings germinated on a medium containing 5 mg L−1 BA revealed increased cell division primarily in the leaf surface tissue on the adaxial side of the mid vein. After the leaves were excised and plated on SIM, these cells continued to divide and differentiate to form adventitious shoots, suggesting that the cytokinin-containing germination medium preconditioned leaves for shoot organogenesis. An in vitro micrografting approach was developed using the elongated shoots as scions, and 57 % of grafted shoots survived to form plants.

Effect of Explant, Genotype and Plant Growth Regulators on Regeneration and Agrobacterium-Mediated Transformation of Potato

A B S T R A C T The objective of the present study is to develop an efficient protocol for plant regeneration and Agrobacterium-mediated transformation of potato cultivars Desiree, Agria and Marfona grown under Iranian agricultural conditions. The regeneration efficiency from internode, leaf and petiole explants was examined. The results from all Shoot Induction Medium (SIM) combinations and all cultivars together indicate highly efficient shoot regeneration from internode (4.56 shoots per explant). The response of petiole and leaf explants was lower, 3.83 and 2.55 shoots per explant, respectively. The highest efficiency of shoot regeneration was achieved with internode explants of cultivar Desiree (6.64 shoots per explant) on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) 1 mg LG 1 + 6-benzyladenine (BAP) 1 mg LG 1 . For plant transformation, internode explants were inoculated and co-cultivated with Agrobacterium tumefaciens strain LBA4404 harboring a binary vector pBI-121 containing β-glucuronidase (GUS) and nptII genes. Reverse transcriptase-polymerase chain reaction analysis and histochemical assay for β-glucuronidase indicated that the gene coding for this enzyme was integrated in the potato genome and could be expressed in potato tissues. The presence of nptII gene in the kanamycin resistant plants was verified by polymerase chain reaction analysis. The transformation frequency ranged from 22-42%.

Direct-shoot organogenesis as an alternative protocol for in vitro regeneration of oil palm (Elaeis guineensis Jacq.)

In the present study, in vitro culture via direct-shoot organogenesis was firstly established as an optional route for clonal propagation in oil palm (Elaeis guineensis Jacq.). The shoot explant excised from zygotic embryo-derived seedling was cultured on half-strength MS (½MS) medium supplemented with two cytokinins, N6-benzyladenine (BA; 8.9, 17.8, 35.5 and 71.0μM) or 6-(γ,γ-dimethylallylamino) purine (2-iP; 9.8, 19.7, 39.4 and 78.7μM). The highest percentage of shoot induction (54.2%) with 5 shoots per explant was obtained when shoot explants were cultured on ½MS medium supplemented with 9.8μM 2-iP. The frequency of direct-shoot organogenesis expressed in the term of percentage of shoot induction was significantly influenced by the duration of incubation period on shoot induction medium. The optimal incubation period of shoot explants on the induction medium (½MS+9.8μM 2-iP) was found to be 4 weeks, which improved the frequency of shoot induction up to 2.6-fold, comparing to those obtained from incubation period of 8 weeks. Histological examination of induced shoots confirmed that newly developed shoot was directly regenerated via direct-shoot organogenesis. Oil palm plants derived from a defined regeneration regime in this study were successfully established in greenhouse. The study established the incubation of 4th week-developmental stage of seedlings as initial explant on shoot induction medium (½MS medium containing 9.8μM 2-iP) with incubation period of 4 weeks as the successful protocol for the direct-shoot organogenesis of oil palm.

Shoot apical meristem ontogeny inArabidopsisembryo explants treated with abscisic acid

The formation of meristemoids and the ontogeny of the shoot apical meristem (SAM) were studied using cultured zygotic embryos of Arabidopsis thaliana (L.) Heynh. LER ecotype. In the callus induction treatment, the procambial cells within the cotyledons of the embryo explants proliferated and gave rise to callus tissues. At the end of the treatment, a band of small cytoplasmic-rich cells derived from the procambium was produced and located at the outer surface of the callus. Upon transfer to the shoot induction medium (SIM) in the absence of abscisic acid (ABA), the cytoplasmic cells differentiated mainly into vascular elements and vacuolated parenchyma cells. This pattern of development negatively affected the explants’ ability to produce meristemoids and SAMs. Contrary to the control, the inclusion of ABA in the SIM resulted first in starch synthesis and accumulation in the surface cytoplasmic cells. This was followed by the formation of cytoplasmic cells among the starch-rich cells; further proliferation of the cytoplasmic cells resulted in the formation of meristemoids. The formation of tracheary elements was suppressed in the ABA-containing SIM. Upon transferring to the shoot development medium, which lacked plant growth regulators, some meristemoids differentiated into apical meristem cells. These cells had distinct nuclei and nucleoli, with little starch present. Additional cell divisions increased the size of the future SAM. Shoot buds with distinct SAMs were clearly delineated with the appearance of leaf primordia.

Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum L. cv. Hezuo 908) with improved efficiency

We developed an efficient and rapid Agrobacterium-mediated tomato transformation protocol by using cotyledon and hypocotyl as explants. The transgenic nature of the regenerants was confirmed through β-glucuronidase activity staining, polymerase chain reaction (PCR), quantitative real-time PCR (qPCR) analysis and northern blot analysis. In the used protocol, the optimized experimental conditions of the tomato genetic transformation were Agrobacterium liquid concentration, equivalent to optical density (OD600) of 1.0, 20 min (cotyledon) or 30 min (hypocotyl) infection time and 2 d of co-culturing period. The optimized medium for shoot induction was Murashige & Skoog (MS) medium + 0.5 mg/L indole-3-acetic acid (IAA) + 0.5 mg/L 6-benzylaminopurine (BAP) for cotyledon and MS + 0.5 mg/L IAA + 2.0 mg/L BAP for hypocotyl. The root induction rate was the highest on the MS medium, supplemented with 0.1 mg/L IAA. The transformation frequency reached 40% per explant. The received results are a base for a larger scale development of transformation procedure.

An Efficient Regeneration System and Optimization of the Transformation from the Cotyledonary Node of Jute (Corchorus capsularis L.)

The purpose of this study was to use cotyledonary nodes as explants to establish an efficient regeneration protocol for jute (Corchorus capsularis L.) on Murashige and Skoog (MS) basal medium. Research into the different growth regulators using the orthogonal design L16 (45) revealed that the best shoot induction medium is MS medium containing 8% (w/v) agar and 3% (w/v) sucrose supplemented with 1.0 mg/L 6BA and 0.25 mg/L NAA. The average number of shoots per explant and the explant induction rate were 9.8 and 100%. After 3 weeks, 2–3 cm shoots were rooting on 1/2 MS medium containing 8% (w/v) agar and 3% (w/v) sucrose supplemented with 1.0 mg/L 6-BA + 0.2 mg/L NAA. Moreover, we optimized Agrobacterium-mediated transformation using the GUS gene transient expression system. The best condition for obtaining higher transformation rate consisted of the use of fresh explants to which 100 μM acetosyringone was added for a co-culture time of 10 min, the OD value of Agrobacterium liquid is 0.5 at 600 nm. These data provide an important basis for the application of other trait gene in the improvement of jute fiber quality.

Abiotic stresses increase plant regeneration ability of rhizome explants of Curcuma caesia Roxb.

Curcuma caesia Roxb., commonly called ''Black turmeric'', is an important medicinal plant. Nor- mally this plant is micropropagated via rhizome or rhizome bud explant culture. However, the shoot induction from rhizome explants is low and thus, alternate methods could be adopted to induce maximum shoots. In this report, we subjected the rhizome segments to abiotic stresses such as warm water, hot air, cold water and sodium chloride (NaCl) at various time intervals for testing their effect on shoot induction. The best shoot induction medium (control) in terms of percent explants producing shoots (66 %) and number of shoots (11.2 per explant) was Murashige and Skoog medium supplemented with 6-thidiazuron (6.8 lM) and indole-3-butyric acid (1.5 lM). The application of various stresses significantly increased both percent re- sponse and number of shoots. Rhizome explants exposed to 1 or 2 h at 30-36 C warm water significantly improved the response. Optimum response (94 % cultures responding with 22.4 shoots per explants) was observed when rhi- zomes were exposed at 33 C for 1 h. In hot air as well as cold water treatments the percent cultures responding and number of shoots showed moderate increase. The increase brought about by 300 and 500 mM NaCl for 1-3 h was significantly higher than control. The rhizomes pretreated with 500 mM NaCl for 3 h showed highest response in contrast to other treatments. Here, 96 % rhizomes re- sponded with an average number of 23.4 shoots per ex- plant. These observations supported the positive role of abiotic stresses in inducing shoots from rhizome segments of C. caesia in cultures.

Establishment of high frequency shoot regeneration system in Himalayan poplar (Populus ciliata Wall. ex Royle) from petiole explants using Thidiazuron cytokinin as plant growth regulator

Populus species are important resources for industry and in scientific study on biological and agricultural systems. Our objective was to enhance the frequency of plant regeneration in Himalayan poplar (Populus ciliata wall. ex Royle). The effect of TDZ alone and in combination with adenine and NAA was studied on the regeneration potential of petiole explants. The explants were excised from Himalayan poplar plants grown in glass-houses. After surface sterilization the explants were cultured on shoot induction medium. High percentage shoot regeneration (86 %) was recorded on MS medium supplemented with 0.004 mg L−1 TDZ and 79.7 mg L−1 adenine. The regenerated shoots for elongation and multiplication were transferred to MS + 0.5 mg L−1 BAP + 0.2 mg L−1 IAA + 0.3 mg L−1 GA3. Root regeneration from shoots developed in vitro was observed on MS medium supplemented with 0.10 mg L−1 IBA. Himalayan poplar plantlets could be produced within 2 months after acclimatization in a sterile mixture of sand and soil. We developed a high efficiency plant regeneration protocol from petiole explants of P. ciliata.

In vitro shoot organogenesis and plant regeneration in Tibouchina aspera Aubl.

TheMelastomataceae is the seventh largest family in the plant kingdom and contains approximately 170 genera and 4600 species widely distributed in tropical and subtropical regions of the world (The Plant List 2015a). Among these, the genus Tibouchina is made up of about 400 species, many native to Brazil but also ranging from Mexico and the West Indies to northern Argentina (Renner 1993; The Plant List 2015b). Many species of Tibouchina are used as ornamental and medicinal plants. Those in the former category serve as bedding or potted plants, while members of the latter category have flavonoids associated with positive health-related attributes (Ciampolini et al. 1995; Yoshida et al. 1999a,b; Dixon et al. 2005) and antifungal properties (Kuster et al. 2009). The flowers of Tibouchina are usually purple and rich in anthocyanins, with a long history as part of the human diet (Bobbio et al. 1985; Janna et al. 2006; Chan et al. 2008). Tibouchina aspera Aubl., which originated in tropical America (Funk et al. 2007), is an evergreen shrub with exotic, fuzzy, lustrous grayish foliage, and acrogenous flowers that appear from early summer to autumn. Although T. asperawas introduced to South China several years ago, it does not produce seed in that climate. In a bid to better exploit and utilize this ornamental and medicinal plant, it is essential to propagate the species. The establishment of an efficient in vitro micropropagation and plant regeneration protocol would allow this objective to be achieved. In this study, shoots were induced in T. aspera from immature leaf explants for the first time. An efficient in vitro propagation and plant regeneration system was established, and flowering of acclimatized plants was achieved. The effects of light versus darkness and of different plant growth regulators (PGRs) in shoot induction media were also investigated.

High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33%), cotyledon (90.11%), leaf (62.96%) and petiole (91.10%) explants on MS medium supplemented with 3.5mg/l BAP + 0.019mg/l NAA 2.5mg/l BAP + 0.5mg/l NAA, 4.0mg/l BAP + 0.5mg/l NAA and 4.5mg/l BAP + 0.019mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10mg/l NAA was found best for root regeneration (100%) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.

Direct plant regeneration from in vitro-derived shoot apical meristems of finger millet (Eleusine coracana (L.) Gaertn.)

A rapid and reproducible protocol for direct plant regeneration has been established using in vitro-derived, actively growing shoot apical meristems (SAMs) of the genotype 'CO(Ra)-14'. Shoot apical meristems from 3-day-old seedlings of three finger millet genotypes viz, 'CO(Ra)-14', 'Try-1', and 'Paiyur-2' were assessed for the efficiency of direct shoot induction. The regeneration efficiency of 'CO(Ra)-14' surpassed the other two genotypes and produced a mass of green, multiple shoots within 12 d on Murashige and Skoog medium containing 17.6 μM 6-benzylaminopurine (BA), 0.9 μM 2,4-dichlorophenoxyacetic acid in combination with 750 mg L−1 proline, 500 mg L−1 casein enzymatic hydrolysate, and 2 mg L−1 glycine. 'CO(Ra)-14' showed a maximum frequency of multiple shoot regeneration (96%) with an average of 8.3 shoots per explant. The age and size of shoot apical meristems played a crucial role in triggering adventitious shoot bud proliferation. The multiple shoot buds elongated spontaneously upon reducing the concentration of BA (to 11.0 μM) in the shoot induction medium. The in vitro proliferated shoots were rooted best in half-strength MS medium containing 2.8 μM indole-3-acetic acid and successfully acclimated in the field, subsequently developing into fertile plants. Thus, we report a short tenure (45 d) in vitro whole plant regeneration system, which could be effectively utilized for the production of transgenic finger millet plants.

Using leaf explants for transformation of Chrysanthemum morifolium ramat. mediated by Agrobacterium tumefaciens

An Agrobacterium-mediated transformation for chrysanthemum using leaf explants of two different cultivars was conducted. Leaves were first infected with Agrobacterium tumefaciens LBA4404 strain, harbouring a binary vector pBI121 carrying the GUS reporter gene and the nptII as the selectable marker gene. 30 seconds for infection of leaves proved to be the optimum duration. Best results were achieved when explants were cocultivated with A. tumefaciens for two days. Shoot induction medium was supplemented with 7 mgl -1 kanamycin and 300 mgl -1 cefotaxime. Putative transgenic plants were obtained after rooting on 1/2 MS medium containing 7mgl-1 kanamycin in the absence of cefotaxime. Transformation efficiency of ‘Reagan Elite Salmon’ (29.1%) was higher than ‘Resomee Splendid’ (9.1%). Presence of the gus gene was verified with histochemical GUS assay and polymerase chain reaction (PCR) of transformed plants.

Influence of Silver Nitrate on Leaflet based Direct Regeneration inArachis hypogaeaL.

Peanut (Arachis hypogaea L.) family Leguminosae is one of the world’s most important oilseed crops. The effect of silver nitrate (AgNO3) on in vitro direct regeneration efficiency of leaflet explants was studied in two cvs ICG 7827 and 13942 of A. hypogaea. This study was conducted to explore the effect of silver nitrate (AgNO3) alone and also in combination with growth regulators, on multiple shoot formation and plantlet establishment from leaflet explants in peanut. The leaflet explants were cultured on shoot induction medium (SIM) containing MS salts with B5 vitamins fortified with AgNO3/BAP/KIN/TDZ alone, AgNO3 in combination with BAP/KIN/TDZ, and AgNO3+BAP+NAA/IAA. When AgNO3 added in combination with BAP+NAA/IAA, enhanced shoot buds induction was found in comparison to other combinations of PGRs used. Maximum average number of shoots (15.04±0.16) per regenerating explant was recorded with 86% of response at 2.0 mg/L AgNO3+10 mg/L BAP+1.0 mg/L NAA in cv ICG 7827 followed by 13.73±0.12 shoots/explant with 83% of response at 2.0 mg/L AgNO3+10 mg/L BAP +0.5 mg/L IAA in cv ICG13942. AgNO3 showed a positive and marked effect on direct enhanced level of multiple shoot induction from leaflet explants. The micro-shoots developed from leaflet explants were elongated on shoot elongation medium (SEM) fortified with 0.5 mg/L BAP. For in vitro rooting the elongated micro-shoots were transferred on to root induction medium (RIM) fortified with NAA/IBA/NAA+IBA and IBA+NAA. Maximum average number of roots (16.28±0.15) per shoot was observed with 90% of response at 1.0 mg/L NAA. The in vitro rooted plantlets were transferred into field. The survival percentage was found to be 90%.

An Efficient Regeneration and Genetic Transformation Protocol ofColeus forskohliiusing Biolistic Gun

An efficient selection and plant regeneration protocol for biolistic gun transformation using leaf derived callus of Coleus forskohlii has been developed. Highest regeneration frequency 90% with 50 shoots per callus clump was obtained on Murashige and Skoog (MS) media supplemented with benzylaminopurine (BAP) 2.0 mg L−1 + naphthalene acetic acid (NAA) 0.5 mg L−1 The rate of shoot multiplication was increased with each subculture. Rhizogenesis was obtained on the same media composition. The in vitro raised plants were established successfully in sand and cocopeat (1:1). Callus of C. forskohlii was bombarded using biolistic gun with pABC plasmid DNA which contains β-glucuronidase (GUS) reporter gene and Arabidopsis thaliana white brown complex homologs (AtWBC19) as selectable marker gene. Kanamycin in the shoot induction medium was compared qualitatively and quantitatively for its efficiency as a selection agent for the selection and regeneration of transgenic plants after biolistic gun transformation. Kanamycin levels at or above 50mg L−1 completely inhibited growth of untransformed shoots. The integration of selectable marker gene GUS and AtWBC 19 into the genome of transgenic plants was confirmed using histoenzymatic GUS assay and polymerase chain reaction (PCR) respectively. These results pave the way for the transformation of Coleus forskohlii with desirable genes.

Sonication assistedAgrobacteriumtransformation of banana cv. Ney Poovan shoot tips with GUS reporter gene

In the present study, Agrobacterium-mediated transformation was performed with shoot tips of banana cultivar, Ney Poovan (AB). The plant expression vector pBI 121, harboring reporter gene, (β-glucuronidase and plant selection marker, kanamycin, was used for co-cultivation. The apical meristems were sonicated and vacuum infiltered with Agrobacterium and co-cultured on liquid MS basal medium supplemented with 100 μM acetosyringone for 72 h. The explants were then cultured on solid shoot induction medium [SIM-MS salts, B5 vitamins, 3% sucrose, 400 mg L1 of cefotaxime and 100 mg L1 of kanamycin and 5 mg L1 of BAP], which showed a regeneration percentage that ranged from 20% to 40%. Among different time intervals tested, sonication assisted Agrobacterium transformation for 8 min was found to be highly efficient in Ney Poovan with a maximum of 56% GUS positive shoots. The uniform GUS staining of transformed plant confirmed the presence and integration of the GUS gene into the banana genome. Polymerase chain reaction confirmed the presence of 500 bp amplified product of partial GUS gene in transformed plants. Thus an efficient protocol for Agrobacterium- mediated transformation of banana cv Ney Poovan was standardized which may possibly be useful for future studies on genetic modification and development of transgenics using novel genes

In vitro regeneration of Cyrtanthus species: ornamental plants with medicinal benefits

Cyrtanthus (Amaryllidaceae) is a genus of perennial geophytes, endemic to the southern African region. Destructive and indiscriminate harvesting of bulbs for medicinal and ornamental purposes has led to intensive decimation of the populations of most of these species in their natural habitats. The aim of this study was to develop in vitro regeneration systems for Cyrtanthus contractus, Cyrtanthus guthrieae, and Cyrtanthus obliquus using twin-scale explants from mature bulbs. Twin scales from the three species were cultured on solid Murashige and Skoog (MS) medium with different concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA) under 16/8-h light/dark conditions at 25 ± 2°C. The best shoot induction responses were obtained on MS medium containing 4.4 μM BA + 1.1 μM NAA (3.1 shoots/explant) for C. contractus and C. guthrieae and 6.7 μM BA + 2.7 μM NAA for C. obliquus. When the best shoot induction medium for each species was investigated for the effect of cytokinins on shoot organogenesis, using different concentrations of BA, kinetin (Kin), meta-topolin (mT), zeatin (ZT), and thidiazuron (TDZ), the medium that produced the best shoot induction for C. guthrieae also produced the highest number of shoots per explant, whereas the highest numbers of shoots per explant were obtained with 10 μM TDZ for C. contractus and 10 μM BA for C. obliquus. Superior quality shoots in all three species were obtained from MS medium supplemented with Kin and mT. Regenerated shoots were rooted successfully on half- and full-strength MS medium without plant growth regulators, transferred to organic soil mix, and successfully acclimatized in the greenhouse. The micropropagation protocols described provide rapid and cost-effective methods for in vitro propagation for application to the conservation and domestication of Cyrtanthus species.