Showing papers on "Kinetin 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.

Detection of somaclonal variation of cotton (Gossypium hirsutum) using cytogenetics, flow cytometry and molecular markers

Abstract: Two protocols of plant regeneration for cotton were adopted in this study, namely, 2, 4-D and kinetin hormone combination and IBA and kinetin hormone combination. Twenty-eight embryogenic cell lines via somatic embryogenesis and 67 regenerated plants from these embryogenic calli were selected and used for random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR), chromosomal number counting, and flow cytometric analysis. The roles of RAPD and SSR markers in detecting somaclonal variation of cotton (Gossypium hirsutum L.) were evaluated. Two cluster analyses were performed to express, in the form of dendrograms, the relationships among the hormone combinations and the genetic variability. Both DNA-based techniques were able to amplify all of the cell clones and regenerated plantlets genomes and relative higher genetic variation could be detected in the culture type with 2, 4-D and kinetin hormone combination. The result suggested that 2, 4-D and kinetin hormone combination could induce relative high somaclonal variation and RAPD and SSR markers are useful in detecting somaclonal variation of regenerated cotton plants via somatic embryogenesis. Chromosome number counting and flow cytometry analysis revealed that the number of chromosomes and ploidy levels were nearly stable in all regenerated plants except two regenerated plantlets (lost 4 and 5 chromosomes, respectively) which meant that cytological changes were not correlated with the frequency of RAPD and SSR polymorphisms. This result also might mean that the cell lines with variation of chromosome numbers were difficult to regenerate plants.

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.

An efficient system for the production of clonal plantlets of the medicinally important aromatic plant: Salvia africana-lutea L.

Abstract: An in vitro cultivation protocol was developed for S. africana-lutea a species threatened by over collection due to its importance as an aromatic medicinal plant in the Western Cape of South Africa. Adventitious shoot induction was most successful using hypocotyls as explants for propagation on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium supplemented with 4.4 μM BA only; 2.7 μM NAA and 4.4 μM BA; or 2.9 μM IAA and 9.3 μM kinetin respectively. For continuous subculture, IAA and BA (μM) at a ratio of 2.9:4.4 or 2.9:8.9 had the best regeneration potential producing approximately three plantlets per nodal explant. Plantlets had 4–5 nodes that could be utilized for the following subculture phase to induce axillary shoots. The tissue culture of S. africana-lutea not only favoured rapid multiplication but was also characterized by seasonal in vitro flowering that was in synchrony with that of plants growing in the wild. This propagation regime has the capacity for producing 2000–3000 plants from one shoot after 3 four-week long subculture cycles, making it highly attractive for implementation as an in vitro conservation strategy. The micropropagated plants were easily acclimatized (88%) within a month after rooting in vitro and planted ex vitro in a sand:soil:peat moss:vermiculite (1:1:1:1; v/v) mixture.

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.

Role of origin and endophyte infection in browning of bud-derived tissue cultures of Scots pine (Pinus sylvestris L.)

Abstract: Callus tissues originating from buds of mature Scots pine (Pinus sylvestris L.) trees exhibit the typical problem of browning, which leads to degeneration and death of the tissues. The effects of medium, origin (tree and location) and endophyte infection were studied on the browning and growth of bud-derived tissue cultures. The calli growing on medium with higher kinetin content and source of organic nitrogen, and originating from the southern location grew better and exhibited less browning. Endophytic microbial cells were detected in the brown callus tissues by transmission electron microscopy. The natural endophyte infection frequency of Scots pine buds was studied and found dependent on the tree, but not on the location. A well-growing, green callus line was artificially infected by an endophytic strain of Methylobacterium extorquens, and browning was not observed on solid media compared to the uninfected control clones of the same callus. However, suspension cultures started from the infected callus died faster than cultures started from the uninfected callus. The endophyte species composition and plant genotype together with tissue culture conditions are the key factors for gaining plant tissue cultures with high regeneration capacity.

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 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).

Phenolic changes during in vitro organogenesis of cotton ( Gossypium hirsutum L.) shoot tips

Abstract: Browning and subsequent death of the cultured explants are major problems for many tissue culture non/hard-adaptive species and usually depended on the phenolic compounds and the quantity of total phenols. Many studies, using shoot tips as explant source indicated various problems such as phenolic exudation, media discoloration, rooting deficiencies and explant browning and death. The phenols are synthesized by the plants and in many tissue culture studies, excreted and then oxidized phenols effect in vitro proliferation negatively. In addition, amount of phenolics can be more or less in different stages of organogenesis due to metabolic actions. Therefore determination of phenolics and calculating the amounts of phenols may be another research area for many tissue culture studies. In this study, total phenol amounts of shoot tip cultures were evaluated during in vitro organogenesis of cotton (Gossypium hirsutum L.). Cotton var. Nazilli 84S was used as explant source and total phenols of young leaves, shoots and the MS (Murashige and Skoog) media (for excreted phenols from explants to medium) were calculated in 7-14-21 and 28 days of culturing period. Seeds were germinated in hormone free MS media in 7 days. After germination, 7 day old meristematic shoot tips were dissected out from seedlings and cultured on MS media, supplemented with 0.1 mg/L Kinetin (KIN). They were grown at 25oC under fluorescent light (7500 lx) 16 h light and 8 h dark for 3 weeks. Singleton-Rossi method based Folin-Ciocaelteu reactive was used for determination of total phenol amount of the young plantlets and it was observed that different parts of the plantlets synthesized more or less amounts of phenolics in different stages of organogenesis. Key words: Cotton, tissue culture, phenolics, browning, organogenesis, shoot tip.

Plant regeneration from callus culture of Curcuma aromatica and in vitro detection of somaclonal variation through cytophotometric analysis

Abstract: Callus cultures initiated from shoot base explants of Curcuma aromatica Salisb. were maintained on Murashige and Skoog (MS) media supplemented with 2 mg dm−3 2,4-dichlorophenoxyacetic acid alone or with 0.5 mg dm−3 kinetin. Plantlets were regenerated from 60 and 180-d-old callus on MS media supplemented with 3 mg dm−3 benzyladenine and 0.5 mg dm−3 α-naphthalene acetic acid. Approximately 8–10 plantlets were produced after 30–40 d of culture per 50 mg of callus inoculated. Out of 113 regenerants analyzed 85 plants were exclusively diploid and 28 were predominantly diploid revealing presence of polyploid nuclei. Frequency of polyploid cells were more in regenerants obtained from 180-d-old callus then from 6-d-old callus which might be attributed to the ageing of callus.

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.

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.

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.

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.

Direct shoot regeneration from leaf explants of Spilanthes acmella

Abstract: Multiple shoots of Spilanthes acmella Murr. were induced from nodal buds of in vivo and in vitro seedlings on Murashige and Skoog (MS) medium containing 1.0 mg dm−3 6-benzyladenine (BA) and 0.1 mg dm−3 α-naphthalene-acetic acid (NAA). Adventitious shoots were successfully regenerated from the leaf explants derived from the above mentioned multiple shoots. The efficiency of shoot regeneration was tested in the MS medium containing BA, kinetin, or 2-isopentenyl adenine in combination with NAA, indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA) and gibberellic acid. Maximum number of shoots per explant (20 ± 0.47) was recorded with 3.0 mg dm−3 BA and 1.0 mg dm−3 IAA. An anatomical study confirmed shoot regeneration via direct organogenesis. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing 1.0 mg dm−3 IBA. 95 % of the plantlets were successfully acclimatized and established in soil. The transplanted plantlets showed normal flowering without any morphological variation.

Micropropagation of Vitex agnus-castus , (Verbenaceae)—a valuable medicinal plant

Abstract: This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA3, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.

Genotype and plant growth regulator-dependent response of somatic embryogenesis from Gentiana spp. leaf explants

Abstract: Gentiana kurroo (Royle), Gentiana cruciata (L.), Gentiana tibetica (King. ex Hook. f.), Gentiana lutea (L.), and Gentiana pannonica (Scop.) leaves derived from axenic shoot culture were used as explants. For culture initiation, leaves from the first and second whorls from the apical dome were dissected and cultured on Murashige and Skoog (MS) basal medium supplemented with three different auxins: 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid (NAA), or 3,6-dichloro-o-anisic acid (dicamba) in concentrations of 0.5, 1.0, or 2.0 mg/l; and five different cytokinins: zeatin, 6-furfurylamonopurine (kinetin), N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ), N-(2-chloro-4-pyridyl)N′-phenylurea, or 6-benzylaminopurine (BAP). The cytokinin concentrations used were dependent on the type of cytokinin and varied between 0.25 and 3.0 mg/l. After 2 mo. of culture, the morphogenic response of explants was assessed. Frequency of embryogenesis was the highest for G. kurroo (54.7%) and dependent on plant growth hormones (PGRs). This gentian was the only species showing morphogenic capabilities on media supplemented with all applied combinations of PGRs, while none of the 189 induction media permutations stimulated somatic embryogenesis from G. lutea explants. G. tibetica and G. cruciata both produced an average of 6.6 somatic embryos per explant, while G. pannonica and G. kurroo regenerated at 15.7 and 14.2 somatic embryos per explant, respectively. Optimum regeneration was achieved in the presence of NAA combined with BAP or TDZ. This auxin also stimulated abundant rhizogenesis. Somatic embryos were also regenerated from adventitious roots of G. kurroo, G. cruciata, and G. pannonica. Somatic embryos converted into plantlets on half strength MS medium.

Effect of growth regulators on rapid micropropagation and psoralen production in Psoralea corylifolia L.

Abstract: A rapid and efficient micropropagation system was developed for Psoralea corylifolia, an endangered, valuable medicinal plant. Multiple shoot buds were obtained in half-strength liquid Phillips–Collins (L2) medium supplemented with 5 μM benzylaminopurine (BA) and 5 μM thidiazuron (TDZ) from apical bud explants of 1-week-old cultures. The shoot buds were subcultured on enriched solid L2 medium supplemented with different concentrations and combinations of BA, kinetin (KIN), 2-isopentenyladenine (2iP), TDZ, bavistin (BVN) and trimethoprim (TMP). Enriched solid L2 medium supplemented with 2 μM BA, 1 μM TDZ and 100 mg l−1 BVN were more effective in producing greater number of shoots per explant (85.2 ± 0.9 shoots/explant) after 4 weeks of culture. The regenerated shoots (40–50 mm in length) rooted and accompanied by hardening upon transfer to 50 μM indole-3-butyric acid (IBA) for 15 min and followed by planting in sterile soil mixture and vermiculate (3:1 v/v), with 50 ml of one-eight strength L2 basal salt solution devoid of sucrose and inositol, supplemented with 5 μM IBA and 100 mg l−1 BVN. The plants achieved 100% rooting with hardening. Subsequently the rooted plants were successfully established in the field. The survival percentage differed with seasonal variations. The concentration of psoralen was evaluated in different tissues of ex vitro and in vivo grown plants by high-performance liquid chromatography (HPLC). Psoralen content was increased in leaves (2.97%), roots (2.38%), stems (5.40%) and seeds (1.63%) of ex vitro plants than the in vivo plants. This system facilitates for commercial and rapid propagation of P. corylifolia for conservation strategies and phytomedicine production.

Conservation of Swertia chirata through direct shoot multiplication from leaf explants

Abstract: Swertia chirata is an endangered gentian species that prefers to grow at higher altitudes. This ethnomedicinal herb is known primarily for its bitter taste caused by the presence of important phytochemicals that are directly associated with human health benefits. Due to a continuous loss of habitat and inherent problems of seed viability and seed germination, alternative strategies for propagation and conservation are urgently required to prevent the possible extinction of this species. We have formulated a reproducible protocol for the rapid propagation and conservation of this plant using leaves taken from in vitro shoot cultures. Direct induction of more than seven shoot buds per explant was achieved for the first time when the explants were placed on MS medium supplemented with 2.22 μM N-6-benzyladenine, 11.6 μM kinetin, and 0.5 μM α-naphthalene acetic acid. Direct organogenesis was noted exclusively from the adaxial surface of the basal segments of leaves. Leaves closer to the apical meristem were more responsive than those farther away from the meristem. Plants raised through direct organogenesis were evaluated for their clonal fidelity by chromosomal analysis and DNA fingerprinting. Complete plants were successfully transferred to the field condition and produced viable seeds. Given the enormous potential of this age-old medicinal plant in terms of potential health-benefitting drugs, this protocol can be used for commercial propagation purposes and to initiate future genetic improvement studies.

Efficient somatic embryogenesis and plant regeneration from shoot apex explants of different Indian genotypes of finger millet (Eleusine coracana (L.) Gaertn.)

Abstract: An efficient somatic embryogenesis and plant regeneration system was developed from shoot apex explants of finger millet, Eleusine coracana Eight genotypes, CO 7, CO 9, CO 13, CO 14, GPU 26, GPU 28, GPU 45, and GPU 48, were assessed in this study The maximum somatic embryogenic induction, at 986%, was obtained from explants cultured on Murashige and Skoog medium supplemented with 180 μM dichlorophenoxyacetic acid and 23 μM kinetin The highest number of shoot induction (26) was observed after transfer of embryonic callus to regeneration medium supplemented with 45 μM thidiazuran and 46 μM kinetin Significant differences were observed between genotypes for somatic embryogenesis and plant regeneration GPU 45 gave the best response, while CO 7 was the least responsive under the culture conditions tested in this study Regenerated plants were successfully rooted and grown to maturity after hardening in soil

Análise de crescimento em plantas de soja tratadas com substâncias reguladoras

Abstract: Growth analysis of soybean plants treated with plant growth regulators This work aimed to verify the effect of plant growth regulators on soybean plant growth and chlorophyll content In an experiment carried out in a greenhouse, soybean plants were cultivated (Glycine max (L) Merrill cv BRS-184) in 10-liter pots containing soil from the arable layer, corrected and fertilized according to the soil analysis The treatments used were: control; GA3 100mgL-1; BAP 100mgL-1; IBA 100mgL-1; Stimulate® (IBA, GA3 and kinetin) 20mLL-1; mepiquat chloride 100mgL-1 and mepiquat chloride 100mgL-1 + BAP 100mgL-1 + IBA 100mgL-1 Treatments were applied three times at 30-day intervals Six samplings were taken at 13-day intervals The results indicated that the highest total dry weight value resulted from the application of IBA and Stimulate®, and that the application of mepiquat chloride in association with IBA and BAP reduced total dry matter production The leaf area was smaller than the control in most treatments The chlorophyll content and growth rate were slightly infl uenced by the treatments The cytokinin treatment alone or in association with other plant growth regulators retained the chlorophyll content RGR and NAR decreased from 99 days after sowing with the application of mepiquat chloride

Callus induction and in vitro plant regeneration of rice (Oryza sativa L.) under various conditions.

Abstract: The objective of the present study was to develop an effective protocol for optimum callus induction and complete plant regeneration for four varieties of rice (Oryza sativa L.) i.e., Super Basmati, Basmati-370, Basmati-371 and Fakhre Malakand. Calli were induced from mature seed scutelum. The Murashige and Skoog (MS) and Chu's N6 media containing hormone 2, 4-D (2, 4-Dichlorophenoxy acetic acid) in different concentrations were used for callus induction. Fakhre Malakand produced maximum calli on N6 media containing 3 mg L(-1) 2,4-D. while other three varieties showed maximum callus induction on N6 media containing 2.5 mg L(-1) 2,4-D. N6 media was found better than MS media for callus induction. For complete plant regeneration the calli of two varieties i.e., Basmati-370 and Basmati-371 were plated on N6 media containing different concentrations of NAA (1-Naphthalene acetic acid) and BAP (6-benzyl aminopurine). The maximum regeneration frequency (%) was observed on N6 media containing NAA 1 mg L(-1) and BAP 2.5 mg L(-1). It took 27-30 days for the callus to regenerate into a complete plant. Basmati-370 produced 4-7 plantlets per callus whereas Basmati-371 produced 4-8 plantlets per callus with regeneration frequencies of 61 and 69%, respectively.

Effects of light and differentiation on gingerol and zingiberene production in callus culture of zingiber officinale rosc.

Abstract: Ginger (Zingiber officinale Rosc.) is a herbaceous, rhizomatous, perennial, which its rhizomes are used as an important spices and medicine all over the world. The aromatic principles include zingiberine, while the pungent principles are known as gingeroles. Callus culture of the plants can produce the same compounds exist on their parent plants. The aim of this work was to evaluate the effect of light and differentiation on gingerol and zingiberine production in callus cultures of Z. officinale. A sterile in vitro plant was prepared by sterilization and subculture of buds, excised aseptically and inoculated into sterile culture jars containing Murashige and Skoog’s (MS) medium and were incubated at 25 ± 2 °C under a 16/8 h light/dark cycle. Then different parts of the sterile plant were inoculated in MS medium supplemented with different concentration of 2,4-dichlorophenoxy acetic acid and kinetin. The jars were separated into two groups, one incubated in dark and the other one in light environment, permanently. Production of metabolites was evaluated by TLC. Some of the metabolites were produced only in presence of light. No gingerol and zingiberen was detected on TLC plates of the dedifferentiated callus grown in light or dark environment. It seems that the production of gingerol and zingiberen in callus culture of Z. officinale is correlated with some sort of differentiation.

Efficient regeneration from hypocotyl explants in three cotton cultivars

Abstract: A high frequency in vitro shoot bud differentiation and multiple shoot production protocol from hypocotyl segments of 8 to 10-d-old seedlings of cotton has been developed. Murashige and Skoog (MS) basal medium with Nitsch and Nitsch vitamins was found to be optimal in shoot regeneration. A combination of 2 mg dm−3 thidiazuron and 0.05 mg dm−3 naphthaleneacetic acid was the most effective for shoot regeneration (76 %) and an average of 10.6 shoots per responding explant. Combination of the cytokinins benzylaminopurine and kinetin induced better regeneration response than their individual treatments. Supplementation of the culture medium with ethylene inhibitor silver nitrate and activated charcoal showed beneficial effects. Optimal rooting was obtained on half-strength MS medium supplemented with 1 mg dm−3 indolebutyric acid and activated charcoal. Scanning electron micrographs of in vitro cultured explants revealed that shoot primordia were formed de novo.

In vitro micropropagation and micrografting of gum Arabic tree [Acacia senegal (L.) Wild].

Abstract: Khalafalla M. M. and Daffalla H. M. 2008. In Vitro Micropropagation and Micrografting of Gum Arabic Tree [Acacia senegal (L.) Wild]. Int. J. Sustain. Crop Prod. 3(1):19-27 In order to find a reproducible method for in vitro multiplication of gum Arabic tree (Acacia senegal (L.) Wild). A protocol for in vitro micropropagatin and micrografting was developed at the laboratory of plant tissue culture, Commission for Biotechnology and Genetic Engineering, Khartoum, Sudan, during the period of October 2006 to October 2007 Multiple shoots were regenerated from cotyledonary node derived from 7-daysold in vitro raised seedlings and nodal segment derived from 12-months-old plant growing in a greenhouse. Explants were cultured on Murashige and Skoog (MS) medium supplemented with 0.5–5.0 mgL -1 of either benzyladenine (BA) or kinetin (Kn) alone or in combination with 0.5 mgL -1 α-naphthalene acetic acid (NAA). The maximum number of shoots per cotyledonary node (8.3 ±0.3) and nodal segment (5.3 ±0.7) explants were obtained on MS medium supplemented with 1.0 mg/l BA after 4 weeks of culture. In vitro regenerated shoots were either rooted in vitro on MS medium supplemented with auxins or micrografted on in vitro induced rootstock. Only 25% of the shoots formed roots after being transferred to MS medium containing 1.0 mgL 1 indole-3-butyric acid (IBA) after 28 days of culture under dark condition.The rate of successfully grafted shoots was influenced by both scion length and rootstock age. 100% successful graft was obtained with scion length of 3.0 cm and rootstock of 14-days-age. In vitro rooted shoots and successful grafts were transplanted to plastic pots containing autoclaved garden soil and sand (3:1), then hardened off and transferred to greenhouse where grown to maturity with 100% success. The success of the micrografting was independent of the nature and concentration of growth regulator used in shoot initiation medium and the time period for induction of shoots. This efficient plant regeneration system provides a solid basis for large scale reforestation and genetic improvement of this important multipurpose leguminous tree.