Showing papers on "Kinetin published in 2001"

Comparative studies of cytokinins on in vitro propagation of Bacopa monniera

Abstract: A mass in vitro propagation system for Bacopa monniera (L) Wettst (Scrophulariaceae), a medicinally important plant, has been developed A range of cytokinins have been investigated for multiple shoot induction with node, internode and leaf explants Of the four cytokinins (6-benzyladenine, thidiazuron, kinetin and 2-isopentenyladenine) tested thidiazuron (68 μM) and 6-benzyladenine (89 μM) proved superior to other treatments Optimum adventitious shoot buds induction occurred at 68 μM thidiazuron where an average of 93 shoot buds were produced in leaf explants after 7 weeks of incubation However, subculture of leaf explants on medium containing 22 μM benzyladenine yielded a higher number (1291) of adventitious shoot buds by the end of third subculture The percentage shoot multiplication (100%) as well as the number of shoots per explant remained the high during the first 3 subculture cycles, facilitating their simultaneous harvest for rooting In vitro derived shoots were elongated on growth regulator-free MS medium and exhibited better rooting response on medium containing 49 μM IBA After a hardening phase of 3 weeks, there was an almost 100% transplantation success in the field

Induction of Highly Embryogenic Calli and Plant Regeneration in Upland (Gossypium hirsutum L.) and Pima (Gossypium barbadense L.) Cottons

Abstract: To accomplish our objective of broadening the number ofregenerable cotton lines, we developed a protocol capable of producing plants through somatic embryogenesis of diverse cotton species. Callus was initiated from hypocotyl and cotyledon explants on a callus initiation medium [CIM; modified MS with 1 mg L -1 kinetin and 2 mg L -1 naphthaleneacetic acid (NAA) 1. Friable embryogenic callus was periodically selected and transferred onto callus selection/maintenance medium (CS/MM) [modified MS with 0.1 mg L -1 kinetin and 0.5 mg L -1 NAA]. The selected callus was then transferred into a liquid embryo initiation medium (EIM) (modified MS medium in which NH 4 NO 3 was removed and KNO 3 amount doubled) followed by transfer to solid embryo maturation media EMMS 2 (0.5 mg L -1 NAA + 0.05 mg L -1 kinetin). The liquid step not only decreased the culturing time but also increased the number of embryos per gram of cultured tissue. Germinating somatic embryos were placed on MS medium with no hormones and plantlets were acclimatized before transfer to the greenhouse. Significant numbers of somatic embryos and their derived plantlets were obtained from a commercial cultivar of G. hirsutum, Deltapine 90 and G. barbadense accession GB-35B126 (PI-528306). The mean embryos per gram for Deltapine 90 on EMMS 2 were higher than those previously reported for Coker 312. Highly significant differences were found between the two genotypes for both embryo and plant production. To our knowledge, this is the first report of regeneration of G. barbadense through somatic embryogenesis.

Plant regeneration from leaf base callus of turmeric and random amplified polymorphic DNA analysis of regenerated plants

Abstract: Callus cultures were initiated from leaf bases of turmeric on Murashige and Skoog's basal medium (MS) supplemented with dicamba, picloram (2 mg l−1) or 1-naphthaleneacetic acid (NAA) (5 mg l−1) in combination with benzyladenine (BA) (0.5 mg l−1). On transfer of callus cultures to medium supplemented with benzyladenine (BA) (5 mg l−1) in combination with triiodebenzoic acid (TIBA) or 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1 mg l−1), green shoot primordia were seen to differentiate from the surface of the callus. On transfer of regenerating cultures to half MS media supplemented with Kn, shoot primordia developed into well developed shoots. When shoots were transferred to medium devoid of phytohormones, complete rooted plants were obtained. Ninety percent of the plants survived to maturity on transfer to soil. Random Amplified Polymorphic DNA (RAPD) analysis of eight regenerated plants using 14 primers when separated on non-denaturing polyacrylamide gels showed 38 novel bands. About 51 bands present in the control were absent in the regenerants. The result indicates that variation at DNA level has occurred during in vitro culture.

Factors affecting in vitro microrhizome production in turmeric

Abstract: In vitro microrhizome production was obtained in turmeric (Curcuma longa Linn.). Freshly sprouted buds with small rhizome portions excised from stored mature rhizomes were cultured on semi-solid culture initiation medium — MS basal medium + 0.88M BAP (6-benzylaminopurine) + 0.92M kinetin + 5% coconut water + 2% sucrose + 0.5% agar — resulting in bud elongation. Multiple shoots were produced from these elongated buds by culturing in liquid shoot multiplication medium — MS basal medium + 2.2 MB AP +0 .92M kinetin + 5% coconut water + 2% sucrose — at 251C and 16-h light (at 11.7mol m 2 s 1 )/8-h dark cycles. Clumps of four to five multiple shoots/single shoots were used in various experiments. Cultures were incubated in the dark at 251C. Half strength MS basal medium supplemented with 80 g l 1 sucrose was found to be optimal for microrhizome production. Cytokinin BAP had an inhibitory effect on microrhizome production. At the highest concentration of BAP tried (35.2M) microrhizome production was totally inhibited. Microrhizome production depended on the size of the multiple shoots used. Microrhizomes produced were of a wide range in size (0.1‐2.0 g) and, readily regenerated when isolated and cultured in vitro on culture initiation medium or shoot multiplication medium. Under in vivo conditions, small (0.1‐0.4 g), medium (0.41‐0.8 g) and big (>0.81 g) microrhizomes regenerated. Plantlets developed from big microrhizomes grew faster.

Effects of auxins and cytokinins on direct somatic embryogenesison leaf explants of Oncidium 'Gower Ramsey’

Abstract: Four auxins (IAA, IBA, NAA and 2,4-D) and five cytokinins (2iP, zeatin,kinetin,BA and TDZ) were examined for their effects on direct somatic embryogenesis onleaf explantsof a sympodial orchid Oncidium 'Gower Ramsey’.On a hormone-freebasal medium, the percentages of embryo formation were 40%, 20%,5% and0% on leaf tips, adaxial sides, wound surfaces and abaxial sides of theleaf explants, respectively, and the average number of embryos per explant was5.6. Embryo formation on leaf explants was retarded by all four auxins tested,but promoted by all the cytokinins. The percentages of embryo formation werereduced to 20%, 5–10%, 0% and 0%,respectively, in the same parts of leaf explants when supplemented with lowdogsages of IAA (0.3–3 mg/l) on the basal medium.Furthermore, embryo formation was totally inhibited by 3 mg/l NAA,0.3–3 mg/l IBA and 2,4-D. The sequence of embryo formationonvarious location of leaf explants was altered by 0.3–1 mg/l2iP and 3 mg/l zeatin, and embryo formation on adaxial sides> leaf tips > woundsurfaces > abaxial sides. The highest percentage of embryoformation on leaf tips, adaxial sides and wound surfaces of explants were75%, 50% and 20% when supplemented with 1mg/lTDZ, 1 mg/l 2iP and 0.3 mg/l kinetin, respectively.The highest average number of embryo per explant (10.7) was found on a basalmedium containing 1 mg/l TDZ.

Regenerated plants in tylophora indica (burm. f. merrill.)

Abstract: embryogenesis is described. Callus induction from leaf explants was on Murashige and Skoog (MS) medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D; 0.0-3 mg 1-1; 0.0-13.56 [LM) and kinetin (Kn; 0.01 mg 1-1; 0.05 pIM). The best response for callus induction was obtained on MS medium containing 2 mg 1-1 (9.04 pM) 2,4-D and 0.01 mg 1-1 (0.05 pM) Kn. After two subcultures on the same medium the embryogenic callus was transferred to MS medium with different concentrations of the cytokinin, 6-benzyladenine (0.5-3 mgl-1; 2.22-13.32 KpM) and 2-isopentenyladenine (2ip; 0.53 mg 1-1; 2.46-14.76 iM) along with 0.01 mg 1-1 (0.05 lM) indole-3-butyric acid (IBA) for somatic embryo development and maturation. MS medium with 2 mg 1-1 (9.84 KM) 2ip produced the maximum number of mature somatic embryos. The mature embryos were bipolar and on transfer to MS basal medium produced complete plantlets. After hardening the regenerants were planted in the Gudalur forests of Western Ghats. Total DNA was extracted from 14 regenerants and the mother plant. Random amplified polymorphic DNA (RAPD) analysis was carried out using 20 arbitrary oligonucleotides. The amplification products were monomorphic among all the plants revealing the genetic homogeneity and true-to-type nature of the regenerants.

Plant regeneration through somatic embryogenesis and rapd analysis of regenerated plants in tylophora indica (burm. f. merrill.)

Abstract: A procedure for the regeneration of complete plantlets of Tylophora indica from cultured leaf callus via somatic embryogenesis is described. Callus induction from leaf explants was on Murashige and Skoog (MS) medium with different concentrations of 2,4-dichlorophenoxyacetic acid (2.4-D; 0.03–3 mg l−1; 0.0–13.56 μM) and kinetin (Kn; 0.01 mg l−1; 0.05 μM). The best response for callus induction was obtained on MS medium containing 2 mg l−1 (9.04 μM) 2.4-D and 0.01 mg l−1 (0.05 μM) Kn. After two subeultures on the same medium the embryogenic callus was transferred to MS medium with different concentrations of the cytokinin, 6-benzyladenine (0.5–3 mg l−1; 2.22–13.32 μM) and 2-isopentenyladenine (2ip; 0.53 mg l−1; 2.46–14.76 μM) along with 0.01 mg l−1 (0.05 μM) indole-3-butyric acid (IBA) for somatic embryo development and maturation. MS medium with 2 mg l−1 (9.84 μM) 2ip produced the maximum number of mature somatic embryos. The mature embryos were bipolar and on transfer to MS basal medium produced complete plantlets. After hardening the regenerants were planted in the Gudalur forests of Western Ghats. Total DNA was extracted from 14 regenerants and the mother plant. Random amplified polymorphic, DNA (RAPD) analysis was carried out using 20 arbitrary oligonucleotides. The amplification products were monomorphic among all the plants revealing the genetic homogeneity and true-to-type nature of the regenerants.

Cytokinin and its precursors for improving growth and yield of rice

Abstract: Phytohormones are well known for their regulatory role in plant growth and development and cytokinins represent one of the five well-established classes of phytohormones. A field experiment was conducted to evaluate the effect of a synthetic cytokinin (kinetin) and its physiological precursors (adenine+isopentyl alcohol) on growth and yield of rice. Five levels (10−2–10−6 M) of each, kinetin and adenine (ADE) plus isopentyl alcohol (IA) were used, in addition to an untreated control. Treatments were applied by dipping roots of rice seedlings in solutions of cytokinin or its precursors for an hour just before transplanting. The precursors were more effective in comparison with pure cytokinin. The plant height was increased by 6.5% over control when treated with 10−5 M ADE+IA. The application of ADE+IA at the rate of 10−4 M significantly increased the number of tillers (34.7%), number of panicles (38.5%) and paddy yield (21.6%) compared to control. Moreover, nutrient (NPK) contents were also significantly improved by 83.1, 36.4 and 26.0% in straw and 53.2, 27.7 and 10.8% in grain, respectively, as compared to control. The findings support the hypothesis that exogenous supply of cytokinin or its precursors in the root zone could improve the growth and yield of treated plant. However, it is difficult to conclude in this study that the precursors were metabolized into cytokinins outside the roots by rhizosphere/rhizoplane microorganisms before being absorbed by the plant roots or converted into cytokinins within the plant tissues. The greater effectiveness of precursors compared to kinetin could be attributed to a gradual conversion of ADE plus IA into cytokinins resulting in a continuous supply over a period of time.

Carbonic Anhydrase, Photosynthesis, and Seed Yield in Mustard Plants Treated with Phytohormones

Abstract: The leaves of 30-d-old plants of Brassica juncea Czern & Coss cv. Varuna were sprayed with 10−6 M aqueous solutions of indole-3-yl-acetic acid (IAA), gibberellic acid (GA3), kinetin (KIN), and abscisic acid (ABA) or 10−8 M of 28-homobrassinolide (HBR). All the phytohormones, except ABA, improved the vegetative growth and seed yield at harvest, compared with those sprayed with deionised water (control). HBR was most prominent in its effect, generating 32, 30, 36, 70, 25, and 29 % higher values for dry mass, chlorophyll content, carbonic anhydrase (E.C. 4.2.1.1) activity, and net photosynthetic rate in 60-d-old plants, pods per plant, and seed yield at harvest, over the control, respectively. The order of response to various hormones was HBR > GA3 > IAA > KIN > control > ABA.

Production of withaferin A in shoot cultures of Withania somnifera.

Abstract: Multiple shoot cultures of Withania somnifera were established from single shoot tip explants and their potential for the production of two principle withanolides, withaferin A and withanolide D was investigated. Shoot tips grown on MS medium supplemented with BA (1 mg l-1) induced 10.0 ± 1.15 microshoots per explants and shoot cultures accumulated both withanolides (withaferin A = 0.04 %, withanolide D = 0.06 %). Supplementation of MSSM (solid) agar medium with 4 % sucrose enhanced accumulation of both withaferin A (0.16 %) and withanolide D (0.08 %). Reduction of the agar concentration to 0.16 % increased the number of microshoots induced per explant to 25.5. MSSM liquid medium containing 10 % coconut milk favoured a maximum increase in biomass (27 fold); number of microshoots induced (37.6 ± 1.45) as well as accumulation of withaferin A (0.14 %). BA:N6-Benzyladenine Kn:Kinetin 2iP:N6-[2-Isopentenyl]adenine MS:Murashige and Skoog (1962) MSSM:Murashige and Skoog's basal medium + BA (1 mg l-1)

Shoot organogenesis and mass propagation of Coleus forskohlii from leaf derived callus

Abstract: A high frequency shoot organogenesis and plant establishment protocol has been developed for Coleus forskohlii from leaf derived callus. Optimal callus was developed from mature leaves on Murashige and Skoog (MS) medium supplemented with 2.4 μM kinetin alone. Shoots were regenerated from the callus on MS medium supplemented with 4.6 μM kinetin and 0.54 μM 1-naphthalene acetic acid. The highest rate of shoot multiplication was achieved at the sixth subculture and more than 150 shoots were produced per callus clump. Regenerated shootlets were rooted spontaneously on half-strength MS medium devoid of growth regulators. The in vitro raised plants were established successfully in soil. The amount of forskolin in in vitroraised plants and wild plants was estimated and found that they produce comparable quantity of forskolin. This in vitro propagation protocol should be useful for conservation as well as mass propagation of this plant.

In vitro regeneration of Acacia mangium via organogenesis

Abstract: Plant regeneration of Acacia mangium was achieved through organogenesis in callus cultures. Calli were induced from five types of explants (embryo axes and cotyledons of mature zygotic embryos as well as leaflets, petioles and stems of seedlings) of A. mangium on MS (Murashige and Skoog, 1962) basal medium containing 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 13.95 μM kinetin (KT). Green or green purple compact nodules containing clusters of meristematic centers were induced in these calli after transfer to MS basal medium containing 1.14–22.75 μM thidiazuron (TDZ) and 1.43–2.86 μM indole-3-acetic acid (IAA). A combination of 4.55 μM TDZ and 1.43 μM IAA promoted the highest percentage of calli to form nodules, in 8–11% of calli derived from cotyledons, embryo axes, leaflets or petiole and in 4% of calli derived from stems. Twenty-two percent of the nodules formed adventitious shoots on MS basal medium containing 0.045 μM TDZ. Shoots were elongated on MS medium containing 0.045 μM TDZ supplemented with 7.22 μM gibberellic acid. The medium containing 10.75 μM NAA and 2.33 μM KT promoted rooting of 10% of the elongated shoots. Plantlets grew up well in the green house.

Effect of various growth regulators on shoot regeneration of sugarcane

Abstract: Sugarcane (Saccharum spp. hybrid cv. CP 84-1198) embryogenic calluses were induced from young leaves cultured on modified Murashige and Skoog basal medium supplemented with 13.6 μM 2,4-dichlorophenoxyacetic acid. Five concentrations, 0.5, 1.0, 2.5, 5.0, and 10.0 μM, of five different growth regulators, 6-benzylaminopurine, kinetin, 6-γ,γ-(dimethylallylamino)purine, zeatin, and thidiazuron, were tested with or without 22.5 μM α-naphthaleneacetic acid to compare their ability to induce regeneration from embryogenic callus. After 4 wk on medium, the percentage of shoot meristem induction was evaluated, and after 10 wk the total number of shoots produced, as well as the percentage of shoots greater than 1 cm in length, was obtained. Although it had the lowest percentage of elongated shoots, medium containing thidiazuron alone performed better than all other growth regulators tested, with the highest percentage of shoot induction and the largest number of shoots, particularly at a concentration of 2.5 μM.

Micropropagation of Rollinia mucosa (JACQ.) baill

Abstract: A protocol for in vitro propagation of Rollinia mucosa, an important medicinal plant, was developed. The presence of 500 mg l−1 polyvinylpyrrolidone (PVP) during explant excision was important to avoid browning. Axillary buds, adventitious buds, and shoot cluster proliferation were achieved from epicotyl and hypocotyl explants from nursery-grown seedlings. The highest direct organogenesis percentage from hypocotyl explants was obtained upon culture of explants on Murashige and Skoog medium supplemented with 2.2 μM benzyladenine (BA) plus 2.32 μM kinetin. Epicotyl explants display highest regeneration frequency on a medium containing 8.8 μM BA and 0.54 μM naphthaleneacetic acid. Gibberellic acid was necessary for shoot elongation. Root induction was observed when shoots were pretreated with activated charcoal for 7 d in the dark before culture on Woody Plant Medium supplemented with 49.21 μM indolebutyric acid for 10 d. Root development was observed when 20 g l−1 sucrose was used. Rooted plantlets were acclimatized and grown in the greenhouse.

High Frequency Somatic Embryogenesis and Plant Regeneration of an Elite Chinese Cotton Variety

Abstract: An elite Chinese cotton (Gossypium hirsutum L) cultivar Simian-3 was chosen for tissue culture Callus with a high frequency of somatic embryogenesis, somatic embryos, and regenerative plants was obtained Callus was induced from three types of explants on MSB (MS salts with B, vitamins) medium supplemented with zeatin (ZT) only, but the percentage of callus induction and growth of callus varied It appeared that it was much easier to induce callus from hypocotyl than cotyledon or root explants The concentrations of ZT were critical to the induction and proliferation of callus The optimum ZT concentration for callus induction was 30~50mg/L Two kinds of callus could be identified after 70 days of culture: embryogenic and nonembryogenic callus Embryogenic callus developed into somatic embryos at various stages after 20 days of subculture The capability of embryogenesis depended on the explant types The root was the most responsive explant for production of somatic embryos, the hypocotyl was the next, and the cotyledon was the last Moreover, a low concentration of ZT was advantageous to the induction of embryogenic callus 2, 4-dichlorophenoxyacetic acid (2, 4-D) promoted the proliferation of embryogenic callus, but had a negative effect on the differentiation and germination of somatic embryos Addition of activated charcoal or a proper combination of ZT and 3-indoleacetic acid (IAA) could promote the production, maturation and germination of somatic embryos The best medium for the proliferation of embryogenic callus was ZH medium (Zhang et al, 1996) with 10mg/L 2, 4-D, 05mg/L kinetin (KT) and 05mg/L ZT The best medium for the differentiation and germination of somatic embryos was MSB with 01mg/L ZT and 2g/L activated charcoal An efficient protocol for the production of high frequency somatic embryogenesis and plant regeneration of an elite cotton variety Simian-3 has been developed Complete plants could be regenerated through somatic embryogenesis from hypocotyl, cotyledon and root explants in 3-4 months

In vitro flowering of bitter melon.

Abstract: Flowers were formed from shoot tips of bitter melon (Momordica charantia L.) cultured on Murashige and Skoog medium supplemented with 90 mM sucrose, 0.05 mM Fe2+ and 4 µM N6-benzyladenine (BA). The addition of 0.05 mM Fe2+ to the medium prevented chlorosis of the explant and promoted normal flowering. Increasing the ratio of carbon to nitrogen promoted male flower formation but intensively inhibited vegetative growth. The influence of cytokinin on the morphogenesis of the explant was highly notable. Flowers could be formed after a 15- to 20-day exposure to kinetin (Kin) or BA. Kin and BA had opposite effects with regard to the development of the explant. Kin promoted flower formation, especially female, but inhibited branch bud formation. Conversely, BA promoted branch bud formation and also promoted male flower formation when present at a concentration of 1–2 µM, but completely inhibited flower formation at 4–8 µM. Fluorescein diacetate staining and in vitro germination showed that in vitro pollen were of a fairly high viability.

Influence of exogenous hormones on growth and secondary metabolite production in hairy root cultures of Cichorium intybus L. cv. Lucknow Local

Abstract: The effect of exogenously fed hormones on hairy root cultures of Cichorium intybus L. ev. Lucknow Local was studied. It was seen that auxin in the presence of low levels of kinetin induces rapid disorganization in hairy root cultures of C. intybus, ultimately to form suspension cultures, and this process was associated with the decrease in coumarin content in the cells. Of various treatments, it was observed that with an increase in the auxin: cytokinin ratio, the biomass decreased with the increase in disorganization index during the culture period of 28 d. The disorganization index was less when the inoculum size was enhanced to 10-fold. The total endogenous indole-3-acetic acid titers and indole-3-acetic acid oxidase activity also decreased with an increase in disorganization index, and was independent of initial inoculum size, with only a magnitude difference. The total coumarin content strictly correlated with growth in all the treatments. In contrast, exogenously supplied gibberellic acid at the 0.5 mg l−1 level enhanced growth, coumarin content, and branching patterns over the control and other treatments on day 28. The exogenously fed growth regulators had an effect on growth, auxin and coumarin biosyntheses, wherein transformed roots treated with increasing concentration of auxin to cytokinin ratios lost their ability for coumarin biosynthesis. The behavior of hairy roots from an Indian cultivar of chicory upon growth regulator treatment is discussed in terms of growth, coumarin and auxin biosyntheses.

In vitro propagation and rhizome formation in Curcuma longa Linn.

Abstract: Turmeric (Curcuma longa Linn.) which is cultivated by underground rhizomes is a slow propagating species. Multiplication and callus induction starting from the rhizome buds and shoot tips of C. longa in MS medium was carried out. A combination of naphthalene acetic acid (NAA; 1.0 mg/l) with kinetin (Kn; 1.0 mg/l) or NAA (1.0 mg/l) with 6-benzylaminopurine (BAP; 2.0 mg/l) was optimum for rapid clonal propagation of turmeric. A concentration of 2.5-3.0 mg/l of 2,4-dichlorophenoxy-acetic acid (2,4-D) was found to be optimum for callus induction. Regeneration of plantlets from a callus was successfully conducted in MS medium supplemented with standard growth hormones for multiplication at 25 +/- 2 degrees C under a 16 h photoperiod. These plantlets were successfully transferred to the field. Plantlets (4-month-old) were incubated in a medium containing different concentrations of sucrose supplemented with NAA (0.1 mg/l) and Kn (1.0 mg/l) at 27 +/- 2 degrees C under an 8 h photoperiod for induction of rhizomes. In vitro rhizome formation was observed in media containing 6 and 8% sucrose.

Micropropagation of Aerides maculosum Lindl. (Orchidaceae)

Abstract: Young leaf segments from plants growing both in vivo and in vitro were cultured on Murashige and Skoog (MS) medium supplemented with auxins [naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D)], cytokinins [kinetin (KN) and N6-benzyladenine (BA)] and coconut liquid endosperm (CW). The explants from mature leaves did not show any growth and turned necrotic, while those obtained from juvenile leaves growing in vitro developed protocorm-like bodies (PLBs) at their cut surfaces within 4–8 wk depending on the growth medium. An optimum of 18 PLBs developed from leaf explants on medium supplemented with 2.0 mg l−1 (8.87 μM) BA. Upon subculture in basal MS medium, the PLBs differentiated into plantlets within 6–8 wk. The resulting plantlets were successfully transferred to vermiculite initially and subsequently to potting mixture; 84% of the plantlets survived after 3 mo. of transplantation.

The effect of cytokinin type and concentration and the number of subcultures on the multiplication rate of some decorative plants

Abstract: Long-term micropropagation of some plants by tissue culture was investigated. The plants studied can be divided into three groups according to their sensitivity to cytokinins benzyladenine (BA) and kinetin (K). (1) Plants (gerbera cultivars) whose multiplication on BA media is not recommended. Permanent high K level must also be avoided. It must be decreased gradually during a long-term culture. After the 8th or 9th subculture the shoots are to be transferred to the resting medium with 0.1 mg/L K and 1.0 mg/L indoleacetic acid. (2) Plants ( Cordyline, Dracaena, Dieffenbachia) that may be multiplied on media containing either BA or K, the latter being preferable as its concentration may be varied within a larger range (0.5-4 mg/L) without negative side effects. (3) Plants ( Philodendron, Spathiphyllum, Musa) that may be multiplied on BA media without even relatively high BA concentrations (5 mg/L) leading to vitrification. However, in order to obtain the best shoots for rooting, the BA content of the multiplication media has to be reduced to 1/2-1/3 of its original value after the 5th or 6th subculture.

In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

Abstract: A high-efficiency two-step culture procedure for direct somaticorganogenesis in loblolly pine (Pinus taeda L.) resulting inthe formation of multiple shoot structures induced on cotyledons andhypocotyls of mature zygotic embryos is described. Mature zygoticembryos of eight genotypes of loblolly pine were used as explants toinduce direct somatic organogenesis with this two-step culture method,involving the induction and the differentiation of direct adventitiousshoots. After mature zygotic embryos of eight genotypes of loblolly pinewere cultured on induction medium containing 2,4-dichlorophenoxyaceticacid (2,4-D) or α-naphthaleneacetic acid (NAA), 6-benzyladenine(BA), and kinetin for 2–3 weeks, embryos were transferred todifferentiation medium. Adventitious shoot regeneration via directsomatic organogenesis with the frequency of 8.7–27.8% wasobtained from mature zygotic embryo cultures of the genotypes tested.The highest mean number of 32.6 adventitious shoots per mature zygoticembryo was produced from genotype La. The tissue culture protocol of invitro shoot regeneration via direct somatic embryogenesis was optimizedafter examining the periods of the induction culture, chillingtreatment, glutamine concentration, and basic medium levels. Rooting wasachieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid(IBA), 0.5 mg/l gibberellic acid (GA3), and 1 mg/l6-benzyladenine (BA), and regenerated plantlets were established insoil. These results suggested that adventitious shoot regeneration viadirect somatic organogenesis could be useful for clonal micropropagationof some genotypes of loblolly pine and for establishing a transformationsystem of this coniferous species.

Factors that affect plant regeneration from in vitro culture of immature seeds in four lentil cultivars

Abstract: An efficient and simple method for plant regeneration from immature lentil seeds (Lens culinaris) is described. Immature seeds from 1 to 6 mm of four lentil cultivars were cultured in vitro on 10 different media. Culture media included different concentrations of N6-benzylaminopurine (BAP), alone or in combination with other phytohormones. After 4 weeks in culture, multiple shoot regeneration was observed using media with BAP. Immature seed size showed significant effect on shoot regeneration. Regenerated shoots (up to 4 shoots per explant on medium with Kinetin (KN) and from 5 to 20 on media with BAP) formed adventitious roots 30 days after transferring them to a medium containing 11.4 μM indole-3-acetic acid (IAA). The efficiency of the rooting medium varied depending upon the shoot-regeneration medium and the cultivar tested. The highest rooting percentage (88.9%) was obtained from regenerated shoots of the cultivar Verdina on a medium with 1 μM α-naphthaleneacetic acid (NAA).

Plant regeneration from embryogenic cultures initiated from mature loblolly pine zygotic embryos

Abstract: Mature zygotic embryos of eight (open-pollinated) families of loblolly pine (Pinus taeda L.) were cultured on eight different basal salt formulations, each supplemented with 36.2 muM 2,4-dichlorophenoxyacetic acid, 17.8 muM 6-benzyladenine, 18.6 muM kinetin, 500 mg l(-1) casein hydrolysate, and 500 mg l(-1) L-glutamine for 9 wk; embryogenic tissue was formed on cotyledons, hypocotyls, and radicles of mature zygotic embryos. Callus was subcultured on the callus, proliferation, medium, the same as the induction medium, bat with one-fifth concentration of auxin and cytokinin for 9 wk. On this medium a white to translucent, glossy, mucilaginous embryogenic callus containing embryogenic suspensor masses (ESMs) was obtained. The highest frequency of explants forming embryogenic tissue, 17%, occurred on a modified Murashige and Skoog salts basal medium containing the concentration of KNO3, Ca(NO3)(2)-4H(2)O, NH4NO3, KCI, ZnSO4-7H(2)O(2), and MnSO4-H2O, 720, 1900, 00 250, 25.8, and 25.35 mg l(-1), respectively. Embryogenic suspension cultures were established by culturing embryogenic callus in liquid callus proliferation medium. Liquid cultures containing ESMs were transferred to medium containing abscisic acid, polyethylene glycols, and activated charcoal for stimulating ther production of cotyledonary somatic embryos. Mature somatic embryos germinated for 4-12 wk on medium containing indole-butyric acid, gibberellic acid, 6-benzyladenine, activated charcoal, and reduced sucrose concentration (15 g l(-1)). Two hundred and ninety-one regenerated plantlets were transferred to a perlite:peatmoss:vermiculite (1:1:1) mixture, then the plants were transplanted to soil in the earth, and 73 plantlets survived in the field.

Compact callus cluster suspension cultures of Catharanthus roseus with enhanced indole alkaloid biosynthesis

Abstract: Compact callus clusters showing a certain level of cellular or tissue differentiation were established from Catharanthus roseus stem and leaf explants in a modified MS liquid induction medium supplemented with 5.37 μM α-naphthaleneacetic acid and 4.65 μM kinetin. In the induction medium most leaf explants developed into friable half-closed hollow callus clusters, whereas in the same medium containing 2,4-dichlorophenoxyacetic acid instead of α-naphthaleneacetic acid, most leaf explants were induced to form dispersed cell suspension cultures. Characteristics of these different types of suspension cultures were compared, and the results showed that the compact callus clusters could synthesize indole alkaloids 1.9- and 2.4-fold higher than the half-closed hollow callus clusters and dispersed cell cultures, respectively. The degree of compaction expressed by the ratio of fresh weight to dry weight of these suspension cultures was correlated to indole alkaloid production. Our studies also postulated that the level of cellular/tissue differentiation might be responsible for these different alkaloid synthesis capabilities. Sucrose regime affected some properties (the size, degree of compaction, differentiation level) of the compact callus cluster cultures and therefore influenced alkaloid production.

Plantlet regeneration from protoplast-derived embryogenic calli of Crocus cancellatus

Abstract: Plant regeneration from protoplast culture of Crocus cancellatus was investigated using regenerable embryogenic calli obtained from shoot meristem culture on LS (Linsmaier and Skoog, 1965) medium containing 4 mg l−1 kinetin and 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D). Protoplasts were isolated directly from embryogenic calli. The best protoplast growth was found on those embedded in Ca-alginate beads and cultured with nurse cells in MS (Murashige and Skoog, 1962) medium supplemented with 2 mg l−1 kinetin, 1 mg l−1 2,4-D and 100 mg l−1 ascorbic acid at 25 °C in darkness. After 4–5 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads, but the protoplasts without immobilization in Ca-alginate beads showed very low cell division. Growth of the microcalli in the medium with nurse cells was much better than in the medium without nurse cells. Transferring beads onto half strength MS medium supplemented with 0.2 mg l−1 kinetin and 0.1 mg l−1 2,4-D, increased the growth of embryogenic calli. Somatic embryo development was observed either on half strength MS medium growth regulator free or with 1 mg l−1 abscisic acid. Matured embryos germinated on half strength MS medium containing 25 mg l−1 of gibberelic acid. Plantlet formation was obtained on half strength MS medium containing 1 mg l−1 6-benzyladenine and 1 mg l−1 α-naphthaleneacetic acid at 20 °C in a 16/8 h light/dark cycle.

In vitro production of plant growth regulators (PGRs) by Azotobacter chroococcum

Abstract: Plant growth regulators like gibberellin, kinetin and Indole acetic acid (IAA) were detected by thin layer chromatography (TLC) and other conventional methods In the culture filterate of Azotobacter chroococcum-strains. Afew strains produced all the three plant growth regulators (PGRS) while others produced either one or two of these. The production of these PGRs was further confirmed by bioassays.