Showing papers on "Murashige and Skoog medium published in 2008"

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

Abstract: A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA3) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.

Protocorm-like body (PLB) formation and plant regeneration from the callus culture of Dendrobium candidum Wall ex Lindl

Abstract: An efficient system was established for a higher frequency of protocorm-like body (PLB) formation from the callus of Dendrobium candidum Wall ex Lindl. The calluses were induced from longitudinally bisected segments of protocorms and subcultured two times every 40d on Murashige and Skoog medium with macronutrients at half strength, micronutrients at full strength, 2% sucrose, and with 8.8μM 6-Benzylaminopurine. PLB formation was achieved when calluses were transferred onto the same basal medium without any plant growth regulators. PLBs developed into intact plantlets about 2cm in height and with four roots when on basal medium with 2.7μM 1-naphthaleneacetic acid. Plantlets were transplanted into vermiculite with a 95% survival rate in a greenhouse. Histological observations proved that globular somatic embryos could be produced from the inside and surface of the embryogenic callus during PLB formation.

Direct organogenesis from leaf explants of Stevia rebaudiana and cultivation in bioreactor

Abstract: Shoot buds were induced directly on either side of midrib from adaxial surface of immature leaf explants in Stevia rebaudiana Bertoni five weeks after culturing in Murashige and Skoog’s nutrient medium supplemented with 8.88 µM of N 6-benzylaminopurine and kinetin ranging from 4.65 to 6.98 µM. Immature leaves of 0.6 to 1 cm were found to produce best response (93 %) with a highest number of 4.93 shoot buds per explant. For elongation of regenerated shoot buds, MS medium supplemented with 30 g dm−3 sucrose and indole-3-butyric acid (IBA) ranging from 4.92 to 7.38 µM were found most suitable. The medium was further modified to suit bioreactor cultivation of regenerated shoots wherein the use of two-fold MS salts and 60 g dm−3 sucrose resulted in a high biomass yield of 50.68 g dm−3 (m/v) accounting for about 590 micro-cuttings in three weeks. Best rooting of micro-cuttings occurred in half strength MS medium supplemented with IBA ranging from 4.92 to 7.38 µM, 15 g dm−3 sucrose and gelled with 0.8 % agar. Rooted plants were successfully established in substrate containing sand, Vermicompost and garden soil in equal proportions and grown in greenhouse. This is the first report on direct shoot regeneration from Stevia leaves.

Use of an adsorbent and antioxidants to reduce the effects of leached phenolics in in vitro plantlet regeneration of faba bean

Abstract: Development of a reliable in vitro regeneration protocol is necessary to facilitate genetic transformation of faba bean. However, leaching of phenolics from the explants of most genotypes of faba bean to the culture medium causes browning, and eventually kills the explants, hindering in vitro regeneration. This study is aimed to minimize the effect of phenolics and to identify the most suitable types of explants for in vitro regeneration. We pre-treated faba bean seeds in polyvinylpyrrolidone (PVP), then cultured different types of explants on tissue culture media supplemented with an adsorbent (activated charcoal) and antioxidants (ascorbic acid, cysteine and silver nitrate). Our results showed that treating the over night soaked seed (after removing the seed coat) with PVP solution (1000 mg/l) for 1 h, followed by culturing in Murashige and Skoog medium (MS medium) with 3% (w/v) sucrose, 0.8% (w/v) agar, 2 mg/l 6-benzylaminopurine and 2 mg/l thidiazuron, supplemented with ascorbic acid (1 mg/l) or activated charcoal (10 g/l), greatly reduced lethal browning in explants and improved shoot regeneration. The shoots rooted on half-strength MS medium supplemented with 0.5 mg/l
-naphthaleneacetic acid. The cotyledonary node is the most suitable type of explant for regeneration. Regenerated plantlets were successfully established in pots and set seeds in the green house.

Response of the cherry rootstock to water stress induced in vitro

Abstract: The in vitro response of sweet cherry (Prunus cerasus × P. canescens) rootstock Gisela 5 to increasing water deficit in the culture medium was studied. Water stress induced by the incorporation of 1, 2 and 4 % polyethylene glycol (PEG-8000) into the Murashige and Skoog medium was applied for 6 weeks. PEG-induced water stress reduced shoot dry mass, length, water content and relative chlorophyll content. Water stress also induced leaf necrosis without causing loss of viability in the explants. The increase in malondialdehyde content indicated oxidative stress. The activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POX) and glutathione reductase (GR) were also significantly elevated. The concentrations of K, Ca, Fe and Mn of shoots were decreased.

In vitro propagation of a multipurpose leguminous tree (Pterocarpus marsupium Roxb.) using nodal explants

Abstract: Pterocarpus marsupium (Bijasal) is a valuable multipurpose forest tree. The regeneration rate in natural habitat is low and the tree is overexploited. An in vitro propagation protocol has been developed from nodal explants obtained from in vitro raised 18-day-old axenic seedlings. The highest shoot regeneration frequency (85%), maximum number of multiple shoots (8.6) as well as length (4.8 cm) were induced from nodal explants on Murashige and Skoog (MS) medium amended with 4.0 μM 6-benzyladenine (BA), 0.5 μM indole-3-acetic acid (IAA) and 20 μM adenine sulphate (AdS). The percentage of shoot multiplication as well as the number of shoots per node remained the same during the first two subculture, afterwards a decline was recorded. Rooting was best induced in microshoots excised from proliferated shoot cultures on semisolid hormone-free half-strength MS medium, after a pulse (dip) treatment for 7 days in half-strength MS liquid medium containing 100 μM indole-3-butyric acid (IBA) and 15.84 μM phloroglucinol (PG). The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 4 weeks before their transfer to a greenhouse, where the established plants showed 75% survival.

Stable genetic transformation of castor ( Ricinus communis L.) via particle gun-mediated gene transfer using embryo axes from mature seeds

Abstract: The first successful attempt to produce stably transformed castor plants through direct gene transfer using particle gun (BioRad) is described. Decotyledonated embryos from mature seeds were germinated and the embryonic axis was induced to proliferate on Murashige and Skoog (MS) medium supplemented with 0.5 mg l−1 thidiazuron (TDZ) and subjected to bombardment after 5–7 days of pre-incubation. The physical parameters for transient transformation were optimized using the UidA gene encoding β-glucuronidase (GUS) as the reporter gene and with hygromycin-phosphotransferase (hptII) gene as selectable marker. Statistical analysis revealed that helium pressure, target distance, osmoticum, microcarrier type and size, DNA quantity, explant type and number of bombardments had significant influence on transformation efficiency, while the effect of genotype was non-significant. Of the different variables evaluated, embryonic axes from mature seeds, a target distance of 6.0 cm, helium pressure of 1,100 psi, 0.6 μm gold microcarriers, single time bombardment and with both pre- and post-osmoticum were found ideal. Selection of putative transformants was done on MS medium supplemented with 0.5 mg l−1 BA and hygromycin (20, 40 and 60 mg l−1) for 3 cycles. The stable integration of the incorporated gene into castor genome was confirmed with PCR and Southern analysis of T0 and T1 plants. Transformation frequency in terms of plants grown to maturity and showing the presence of the introduced genes was 1.4%. The present results demonstrate the possibility of transformation of embryonic meristematic tissues of castor through particle delivery system.

An efficient protocol for large scale production of sugarcane through micropropagation

Abstract: A rapid propagation and acclimatization response of two different varieties of sugarcane (CP 77,400 and BL-4) was obtained in this study. The shoot apical meristem of different sizes was cultured on MS medium supplemented with different concentrations and combinations of BAP and kinetin either alone or in combination with each other or GA3. Best shoot formation response in CP 77,400 was obtained on MS medium containing 1.5mg/l BAP while in BL-4 the combination of 0.5 mg/l BAP with 0.25 mg/l Kinetin showed best shoot formation response from apical meristem. Meristem of 3.0 mm size proved to be the best size for micropropagation of sugarcane. Excellent multiplication response of In vitro formed shoots was obtained when the concentration of BAP was decreased to 1.0 mg/l in CP 77, 400 and 0.25 mg/l BAP & Kin in BL-4 (i.e. 0.25 mg/lBAP + 0.25 mg/l Kinetin. MS medium containing 1.0 mg/l NAA and 2.0 mg/l IBA showed 100% rooting response of In vitro regenerated shoots of both the varieties of sugarcane within eight days of inoculation. Best hardening response was obtained in Sand+ Soil + Peat (1:1:1) after three week of transplantation in glass house.

Rapid in vitro multiplication and plant regeneration from nodal explants of Andrographis paniculata: a valuable medicinal plant

Abstract: A rapid and efficient method for the large-scale propagation of a highly valuable medicinal plant, Andrographis paniculata Nees, through in vitro culture of nodal explants obtained from 15-d-old aseptic seedling has been developed. High frequency direct shoot proliferation was induced in nodal explants cultured on Murashige and Skoog’s medium supplemented with 6-benzylaminopurine (BAP). Amongst the various cytokinins tested (BAP, kinetin, thidiazuron and 2-isopentyl adenine), BAP proved to be the most effective. The shoot forming capacity of the nodal explants was influenced by the BAP concentration (1–12.5 μM), and the optimal response was observed at 10 μM BAP, which induced an average of 34 shoots in 94% of the cultures within 4 wk. Significant differences were recorded in terms of average number of shoots per explant (8.6–34.1) among the different concentrations of BAP investigated. Concentrations of all cytokinins tested reach a level that can be considered above the optimum level, as marked by a reduced frequency of shoot proliferation. The multiple shoots obtained on various concentrations of BAP failed to elongate even after transfer to hormone-free MS medium. Elongation of the induced shoots was achieved on MS basal medium supplemented with 1.0 μM GA3 within 2 wk. A proliferating shoot culture was established by repeatedly subculturing the original nodal explants on shoot multiplication medium after each harvest of the newly formed shoots. The explants retained their morphogenic potential even after three harvests. Therefore, in 90 d, about 60–70 shoots were obtained from a single nodal explant and the nodal explants from primary shoots further regenerated equivalent number of shoots, depicting their high frequency regeneration potential in A. paniculata. Rooting was best induced in 94% of shoots cultured on MS medium supplemented with 2.5 μM indole-3-butyric acid (IBA), within a wk. The plantlets were successfully transferred to soil after hardening with a 92% survival rate. The system is rapid: the initiation of shoot buds to the transplanting of regenerants to soil is completed in 8–9 wk.

Plant regeneration via protocorm-like body formation and shoot multiplication from seed-derived callus of a maudiae type slipper orchid

Abstract: Tiny seeds from 5-month-old green capsules of a maudiae type slipper orchid, Paphiopedilum Alma Gavaert, were induced to form totipotent callus on 1/2 strength MS medium supplemented with 22.60 μM 2,4-D and 4.54 μM TDZ in darkness. The callus was proliferated more and maintained without any morphogenesis on the same medium with a 2-month interval of subculture for more than 2 years. When transferred to 1/2 MS medium supplemented with 26.85 μM NAA, an average of 4.7 protocorm-like bodies (PLBs)/shoot buds formed from each explant after 120 days of culture. After another 72 and 240 days of culture on the same medium, 25 shoot buds and eventually 75 plantlets were obtained through shoot multiplication from the original culture. Kinetin at 4.65 μM was suitable for shoot multiplication and could induce an average of 3.0 shoots from a single young shoot after 60 days of culture. The regenerated plantlets grew normally when transplanted to containers with sphagnum moss in a shaded greenhouse.

Micropropagation of Dendrobium densiflorum Lindl. ex Wall. through protocorm-like bodies: effects of plant growth regulators and lanthanoids

Abstract: An efficient microprogation protocol has been developed for Dendrobiumdensiflorum Lindl. ex Wall., a traditional medicinal plant, through protocorm-like bodies (PLBs) from nodal stem segments using 6-benzylamino-purine (BAP) and the lanthanoid neodymium. The highest percentage of explants producing PLBs (72%), with an average of 15 PLBs per explant, was induced by culturing stem segments on Murashige and Skoog (MS) medium supplemented with 5.0 mg l−1 BAP. The newly formed PLBs proliferated well on the basal MS medium and completely converted into shoots on MS medium containing 2.0 mg l−1 BAP. Shoots produced an average of 22 roots per plantlet when cultured on MS medium supplemented with 2.0 mg l−1 neodymium nitrate. Healthy plantlets with well-developed roots were successfully acclimatized. The obtained result suggests that the lanthanoids can be used to effectively initiate rooting in the micropropagation and conservation of D. densiflorum.

Micropropagation of gerbera: lipid peroxidation and antioxidant enzyme activities during acclimatization process

Abstract: Gerbera jamesonii H. Bolus ex Hook (Family: Asteraceae) has been successfully acclimatized from temperate to subtropical North Indian plains of Lucknow through in vitro propagation. Flower heads were collected from greenhouse, segmented into 4–16 pieces and cultured in Murashige and Skoog’s medium (MS) (Physiol Plant 15:472–497, 1962) supplemented with 2.87 μM indole-3-acetic acid (IAA) and 8.88 μM N6-benzyladenine (BA) for shoot regeneration. Shoots were subcultured on growth regulator free MS medium. Apical shoot meristems from in vitro plantlets of gerbera were tested in MS medium with different combination of cytokinins [BA, kinetin, and thidiazuron (TDZ)] alongwith 2.68 μM 1-naphthaleneacetic acid (NAA) for shoot multiplication. The optimum results were obtained with 8.88 μM BA. Regenerated plants with well-established root system were transferred to pots containing soil and sand (1:1 v/v) and were kept in humidity chamber with 80–90% relative humidity for 0, 5, 10, 15, 20, and 25 days before they were transferred to field (during October, 2005 to February, 2006). Survival percentage was higher when regenerated plantlets were kept under humidity chamber for 15 days. An attempt was made to obtain basic information on different biochemical changes during acclimatization process of in vitro raised plantlets. Increased lipid peroxidation and high H2O2 content in early stages of acclimatization process reflected a similar process of oxidative stress. Our work suggests that tissue-cultured plants develop antioxidant enzymatic protective system which determine the ability to survive in oxidative stress and up regulation of these enzymes would help to reduce the built up of reactive oxygen species (ROS).

Plant growth regulators affecting in vitro cultivation of Stevia rebaudiana

Abstract: In order to maximize efficiency of plant propagation via direct organogenesis, the influence of plant growth regulators on the growth and development of Stevia rebaudiana grown in vitro was studied. Results indicated that benzyl adenine increased multiplication rate, vitrification and somaclonal variation. However, the best results were recorded with MS nutrient medium without plant growth regulators during in vitro growth and development of Stevia rebaudiana. MS basal medium supplemented with 2 mg/l BA recorded the highest number of shoots, but these shoots were very thin and vitrified and not suitable for multiplication through several subcultures. The nutrient medium (MS basal medium) supplemented with 10 mg/l kinetin recorded 45 shoots / explant as compared to MS nutrient medium which recorded 6.63 shoots / explant but growth parameters for Stevia rebaudiana plantlets grown in MS medium without kinetic is better than MS medium containing kinetin at high concentrations. Nutrient medium (MS basal medium) supplemented with IBA at low concentrations (0.01mg/l) or without auxins achieved the best in vitro growth of Stevia rebaudiana plantlets (100 % root formation). IBA was better than NAA and IAA for shoot and root formation. Increasing NAA concentrations decreased gradually number of shoots. Regarding the effect of NAA on root formation, data indicated that the per cent of shoots formed roots was 87% on MS basal medium without plant growth regulators as compared to MS basal medium supplemented with NAA at low concentrations 0.001, 0.01 and 0.1 mg/l NAA where root per cent was 80%, 73 % and 53 % respectively. On the other hand, NAA at 1.0 and 1.5 mg/l did not help shoots to form roots. Statistical analysis of variance showed no significant differences amongst IAA treatments for in vitro growth of plantlets. Stevia rebaudiana plants were adapted and grown well in planting media containing peat moss, sand and vermiculite at equal volume.

Importance of co-cultivation medium pH for successful Agrobacterium -mediated transformation of Lilium × formolongi

Abstract: An efficient system for Agrobacterium-mediated transformation of Lilium × formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 μM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.

Agrobacterium rhizogenes mediated transformation of Scutellaria baicalensis and production of flavonoids in hairy roots

Abstract: Using different explants of in vitro seed grown Scutellaria baicalensis Georgi plantlets, hairy roots were induced following inoculation of Agrobacterium rhizogenes strains A4GUS, R1000 LBA 9402 and ATCC11325. The A4GUS proved to be more competent than other strains and the highest transformation rates were observed in cotyledonary leaf explant (42.6 %). The transformed roots appeared after 15–20 d of incubation on hormone free Murashige and Skoog medium. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Maximum growth rate was recorded in root:medium ratio 1:100 (m/v). Hairy root lines were further established in Gamborg B5 medium and the biomass increase was maximum from 15 to 30 d. PCR, Southern hybridization and RT-PCR confirmed integration and expression of left and right termini-linked Ri T-DNA fragment of the Ri plasmid from A4GUS into the genome of Scutellaria baicalensis hairy roots. GUS assay was also performed for further integration and expression. All the clones showed higher growth rate them non-transformed root and accumulated considerable amounts of the root-specific flavonoids. Baicalin content was 14.1–30.0 % of dry root mass which was significantly higher then that of control field grown roots (18 %). The wogonin content varies from 0.08 to 0.18 % among the hairy root clones which was also higher than in non-transformed roots (0.07 %).

High in vitro production of ant-canceric indole alkaloids from periwinkle ( Catharanthus roseus) tissue culture

Abstract: Periwinkle (Catharanthus roseus) is one of the most important medical and ornamental plants in the world. In this investigation, periwinkle seeds, after sterilization were cultured on MS medium. Petiole segments of seedlings (4 day old) were subcultured to medium containing various concentrations of NAA accompanied with Kin and subcultured to regenerate the callus and root. Callus and roots were obtained from petioles in some of treatments. The extracts of callus and roots from different treatments were analyzed by spectrophotometer, TLC and HPLC with respect to the indole alkaloids producing capacity. Alkaloids were produced callus and roots from petiole of C. roseus in the presence of 0.1, 5, 10 and 20 mg/l Kin and NAA. MS with 0.1 mg/l NAA + 0.1 mg/l Kin had the highest vindoline, catharanthine, vincristine and root organogenesis capacity. But the level of these alkaloids and ajmalicine were very low compared to that in petiole of intact plant, and the level of serpentine was similar. New roots, callus roots, and callus from MS medium containing 0.1 mg/l NAA + 0.1 mg/l Kin were subcultured in hormone-free and 0.1 mg/l NAA + 0.1 mg/l Kin media and for organogenesis and growth. The most alkaloids amount was produced in new roots and callus roots. The indole alkaloid levels of new roots in new media were higher than in petioles of intact plants. In this study, 10-fold catharanthine, 125-fold serpentine, 0.5-fold vindoline and 0.34-fold ajmalicine were produced by new roots. The most interesting result was presentation of two important ant-cancer dimeric alkaloids, vinblastine and vincristine with amounts of 20-fold vinblastine and 6-fold vincristine to compare that in the petioles of intact plants.

Effect of genotype, explants, growth regulators and sugars on callus induction in cotton (Gossypium hirsutum L.)

Abstract: Ten cotton (Gossypium hirsutum L) genotypes were chosen for tissue culture Callus initiation was genotype dependent, and R405-2000 has the best callogenesis response Callus was induced from three media, the percentage of callus induction and dry weight of callus varied, but MS was the best callogenesis medium It appeared that it was much easier to induce callus from hypocotyl than cotyledon or root explants Induction callus of cotton was varied with hormone regimes In effect, a proper combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) promoted the callus initiation Glucose was the best sugar to promote the production of callus However, the concentrations of glucose were critical to the induction of callus The optimum glucose concentration for callus induction was 40 g/L The best medium for the proliferation of callus was MS medium with 01 mg/l 2,4-D, 05 mg/l KIN and 4% glucose An efficient protocol for the production of high frequency callus of cotton has been developed

In vitro propagation of a high value medicinal plant: Asparagus racemosus Willd.

Abstract: Asparagus racemosus Willd. is an important medicinal plant of tropical and subtropical India. Its medicinal usage has been reported in the Indian and British Pharmacopoeias and in traditional systems of medicine such as Ayurveda, Unani, and Siddha. The multiple uses of this species have increased its commercial demand, resulting in over-exploitation. Because of destructive harvesting, the natural population of A. racemosus is rapidly disappearing, and it is recognized as ‘vulnerable’ (Warner et al., Some important medicinal plants of the Western Ghats, India: a profile. International Development Research Centre, Artstock, New Delhi, India, 15 pp, 2001). The development of an efficient micropropagation protocol will play a significant role in meeting the requirements for commercial cultivation, thereby conserving the species in its natural habitat. In the present study, in vitro shoot proliferation was obtained by culturing single node segments in Murashige and Skoog’s (MS) medium supplemented with 3.69 µM 2-isopentyl adenine and 3% sucrose with a multiplication rate of 3.5. For proper root formation, the in vitro-formed shoot clusters were cultured on half strength (major salts reduced to half) MS medium with 1.61 µM 1-naphthalene acetic acid, 0.46 µM kinetin, 98.91 µM adenine sulfate, 500 mg/l malt extract, 198.25 µM phloroglucinol, and 3% sucrose. On this medium, 85% rooting was observed within 20 d. Following a simple hardening procedure involving sequential transfer of plants to a greenhouse, polyhouse, and shade net, the tissue-cultured plants were transferred to the field where the survival rate was 100%.

An efficient protocol for in vitro propagation of carnation (dianthus caryophyllus)

Abstract: The present research work involves shoot formation, their multiplication and rooting in carnation Dianthus caryophyllous. For shoot formation both apical and nodal meristems were used. MS medium containing BAP alone or in combination with kinetin was tested. Best shoot formation response was obtained after 6 days of inoculation from apical meristem and after 7 days of inoculation from nodal meristem on MS medium supplemented with 4.0 mg/l BAP. Apical meristem showed more pronounced effect for shoot formation than nodal meristem. Well-developed shoots were shifted for their multiplication. Maximum number of multiple shoots were obtained on MS medium containing 1.0 mg/l BAP. These multiple shoots increased in their number when were given subsequent incubation period. Addition of Kinetin to BAP failed to show good shoot multiplication response. Shoots after attaining the size of 5.0 cm were shifted for rooting. Best rooting response was obtained on MS medium containing 1.0 mg/l NAA. Well rooted plants were shifted into glass house for hardening and acclimatization and were shifted to natural climatic conditions.

An improved protocol for Agrobacterium -mediated transformation of grapevine ( Vitis vinifera L.)

Abstract: An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.

Adventitious root induction in Ophiorrhiza prostrata: a tool for the production of camptothecin (an anticancer drug) and rapid propagation

Abstract: Roots of Ophiorrhiza prostrata D. Don serve as a rich source of camptothecin (CPT), an anticancer drug. Because of the large-scale collection of its roots, the plant has become a threatened species. The present study accomplishes the induction of adventitious roots as a means for the production of CPT as well as for the large-scale propagation of this anticancer drug plant using leaf and internode explants. The biomass yield and CPT content of adventitious roots induced from different explants were compared to roots developed on ex vitro rooted stem cuttings. Adventitious roots were produced on half-strength Murashige and Skoog (MS) medium supplemented with 10.74 μM α-naphthaleneacetic acid and 2.32 μM kinetin at mean fresh weights of 0.753, 0.739 and 0.748 g roots from leaf, internode and shoot, respectively. CPT yield from in vitro derived roots after 50, 80 and 120 days of incubation (0.028, 0.06 and 0.1% dry weight, respectively) was not significantly different from those harvested at the same age from ex vitro rooted (0.03, 0.06 and 0.13%, respectively) stem cuttings. CPT from subcultured roots derived from solid (0.08%) medium was lower than from suspension culture medium (0.12%). Subsequent cultures of the adventitious roots showed a stable production of CPT (0.16%). The yield of CPT from 360-day-old plant-derived roots was 0.19%. Elicitation using methyl jasmonate and acetyl salicylic acid exhibited no enhancement in CPT yield. In vitro propagation through direct shoot regeneration was achieved from the adventitious roots upon transfer to MS medium with 8.87 μM N6-benzyladenine (BA) and 2.46 μM indole-3-butyric acid (IBA) with a mean of 21.2 shoots per culture in 50 days. The shoots upon subculture on medium having the same level of BA and IBA underwent rapid proliferation. The shoots transferred to field conditions after in vitro rooting exhibited 95% survival. Adventitious root induction, from leaf and internode explants, enables the feasible production of CPT as well as the large-scale rapid propagation of this species which can safeguard it from extinction.

Factors affecting maintenance, proliferation, and germination of secondary somatic embryos of Eucalyptus globulus Labill

Abstract: The described protocol for repetitive somatic embryogenesis (SE) in Eucalyptus globulus produced more somatic embryos than the primary SE protocol. Primary somatic embryos (induced on MS3NAA) were transferred to the same medium, leading to new cycles of somatic embryos, for at least 2 years. The influence of medium (MS and B5), plant growth regulators (auxins and cytokinins), and light on secondary SE was tested. The MS medium without growth regulators (MSWH) was more efficient for cotyledonary embryo formation and germination than the B5 medium. Reducing auxin (NAA) levels increased the proliferation of globular somatic embryos and allowed SE competence to be maintained on medium free of plant growth regulators. The addition of two cytokinins (BAP and KIN) to the MS medium did not improve proliferation of globular secondary embryos, but was crucial during later stages of the SE process (germination and conversion). Data also show that light may influence the quality of the process, depending on its stage. Darkness should be maintained until the cotyledonary stage is reached, after which exposure to light is recommended.

Optimization of mature embryo-based high frequency callus induction and plant regeneration from elite wheat cultivars grown in China

Abstract: We have developed an efficient procedure for plant regeneration of elite wheat cultivars using mature embryos. Firstly, we established the optimal combination of basal media, inoculation method and pretreatment method using biostatistical methods. The results indicated that the combination of MS medium and longitudinally bisected mature embryos showed the highest culture efficiency, whereas the pretreatment method had no significant effects on callus induction or plant regeneration. A 70% primary callus induction rate was achieved on MS medium containing 2 mg/l 2,4-D for all tested cultivars. Primary calli were then transferred onto the subculture medium to initiate embryogenic calli. Supplementation of the subculture medium with the appropriate combination of phytohormones (2.0 mg/l 2,4-D, 0.5 mg/l BA and 0.1 mg/l NAA) significantly enhanced embryogenic callus production. The addition of AgNO 3 (10 mg/l) in regeneration medium promoted plant regeneration, whereas CuSO 4 stimulated root formation. The use of this protocol achieved successful plant regeneration in eight tested cultivars. The culture efficiency ranged from 15.3% to 36.8%, suggesting this regeneration system may be an effective alternative for wheat genetic transformation.

An improved plant regeneration system and ex vitro acclimatization of Ocimum basilicum L.

Abstract: An efficient, rapid and large scale propagation of a multipurpose herb, Ocimum basilicum through in vitro culture of nodal segments with axillary buds from mature plants has been accomplished. Among the cytokinins, 6-benzyladenine (BA), thidiazuron (TDZ), kinetin (Kin) and 2-isopentenyl adenine (2-iP) tested as supplements to Murashige and Skoog (MS) medium, 5.0 μM BA was optimum in inducing bud break. The highest rate of shoot multiplication was achieved on half-strength MS medium supplemented with 2.5 μM BA and 0.5 μM indole-3-acetic acid (IAA) combination. The shoots regenerated from TDZ supplemented medium when subcultured to hormone-free MS medium considerably increased the rate of shoot multiplication and shoot length by the end of third subculture. For rooting, MS medium supplemented with 1.0 μM indole-3-butyric acid (IBA) proved to be better than that supplemented with IAA or α-naphthalene acetic acid (NAA). The in vitro raised plantlets with well developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 90% survival rate. Chlorophyll a and b, carotenoids and net photosynthetic rate were measured in leaves during ex vitro acclimatization at 0, 7, 14, 21 and 28 days. Firstly these parameters showed a decreasing trend but subsequently increased after 7 days of acclimatization. These findings indicate that the adaptation of micropropagated plants to ex vitro conditions is more extended in time than generally accepted.