Effect of cytokinin types, concentrations and their interactions on in vitro shoot regeneration of Chlorophytum borivilianum Sant. & Fernandez

Background Chlorophytum borivilianum is a rare medicinal plant originally distributed throughout the forest of India. The tubers of C. borivilianum are used as an aphrodisiac and impotence supplement. The propagation of C. borivilianum is possible through seeds and tubers, but conventional methods may take several months. Hence in vitro technique of shoot regeneration could be an efficient alternative means of propagating the species. Latest study reported microtuberization of C. borivilianum but there is no sufficient study on a rapid method for shoot multiplication and elongation. Results Young shoot buds of C. borivilianum were cultured on MS medium containing 6-benzylaminopurine (BAP) and Kinetin (Kn), both at 0, 8.88, 17.8 and 26.6 µM, either individually or in combinations. Proliferated shoots were subcultured on fresh medium of the same constituents on week 3 of culture for further shoot multiplication and elongation. BAP alone (8.88-26.6 µM) was significantly effective on shoot multiplication, while Kn alone (8.88-26.6 µM) was significantly effective on shoot elongation compared to the control containing MS basal medium without any plant growth regulator. However, combination of both cytokinins stimulated an interaction producing higher shoot number and shoot length compared to their individual application. Conclusions The most suitable combination was 8.88 µM BAP + 8.88 µM Kn, reaching a mean shoot number of 10.83 and shoot length of 6.85 cm.

Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll.

Entamoeba histolytica contains a novel calcium-binding protein like calmodulin,which was discovered earlier,and we have reported the presence of its homologue(s)and a dependent protein kinase in plants.To understand the functions of these in plants,a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP)was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised.These plants showed variation in several phenotypic characters,of which two distinct features,more greenness and leaf thickness,were inherited in subsequent generations.The increase in the level of total chlorophyll in different plants ranged from 60% to 70%.There was no major change in chloroplast structure and in the protein level of D1,D2,LHCP and RuBP carboxylase.These morphological changes were not seen in antisense calmodulin transgenic tobacco plants,nor was the calmodulin level altered in EhCaBP antisense plants.

Reduction of vitrification in in vitro raised shoots of Chlorophytum borivilianum Sant. & Fernand., a rare potent medicinal herb.

Reduction of vitrification in in vitro raised shoots derived from shoot bases and immature floral buds along with inflorescence axis used as explants of C. borivilianum, a rare medicinal herb is described. Shoot multiplication was obtained on MS medium with 2 mg l(-1) benzylaminopurine (BAP) + 0.1 mg l(-1) indole-3-butyric acid (IBA) and MS medium with 2 mg l(-1) kinetin (Kin) + 0.1 mg l(-1) 2,4-dichlorophenoxy acetic acid (2,4-D) from shoot bases and inflorescence axis respectively. Best multiplication rates were obtained from both the explants on MS medium with 2 mg l(-1) BAP. Vitrification of shoots in cultures appeared during the multiplication stage. Culture bottles with aerated caps reduced the vitrification to 80%. Reduction of BAP concentration from 2 mg l(-1) to zero during subsequent subcultures also minimized vitrification. Use of 0.5-2 mg l(-1) Kin produced healthy shoots when compared to BAP. In vitro raised shoots rooted on Knop salts containing iron and vitamins of MS medium, 2 mg l(-1) IBA and 0.1% activated charcoal. About 80% plantlets survived upon soil transfer. Scanning electron microscopic and image analyzer studies reveal the morphological structural differences between the leaves of normal and vitrified plantlets.

Influence of cytokinins, auxins and polyamines on in vitro mass multiplication of cotton (Gossypium hirsutum L. cv. SVPR2).

In the present investigation, the influence of different forms of cytokinins, auxins and polyamines were tested for mass multiplication and regeneration of cotton. Initially, for the identification of effective concentration for multiple shoot induction, various concentrations of BAP, Kin and 2iP along with IAA and NAA were tested. Among tested concentrations, media fortified with MS salts; B5 vitamins; 30 g/l, glucose; 2.0 mg/l, 2iP; 2.0 mg/l, IAA and 0.7 % agar showed best response for multiplication of shoot tip explants (20 shoots per shoot tip explants). In nodal explants, maximum of 18.6 shoots were obtained in the media fortified with MS salts, B5 vitamins, 30 g/l, glucose, 2.0 mg/l, 2iP, 1.0 mg/l, NAA and 0.7 % agar. Effect of different concentrations of polyamines like spermidine and putrescine were also tested along with the above said multiplication media. Among the various treatments, 20 mg/l of putrescine showed best response and the multiple of shoots were increased to 26.5 shoots per shoot tip explants and 24.5 shoots per nodal explants. Elongation of shoots was achieved on multiple shoot induction medium. Significant number of roots were initiated in the medium supplemented with MS salts, vitamin B5 and IBA (2.0 mg/l). The frequency of root induction was increased by addition of, PVP (10 mg/l) along with root induction medium and after 2 weeks, the roots reached the maximum length of 22 cm. Further, these plantlets were hardened by using sand, soil and vermiculate in 1:1:1 ratio. The hardened plants were transferred to the environmental growth chamber for proper acclimatization. The hardened plants were then transferred to field for boll yielding and they exhibited 100% survival.

Cytokinin oxidase activity and cytokinin content in roots of sunflower under water stress.

Contents of trans-zeatin riboside (ZR), dihydrozeatin riboside (DZR) and N6-(delta2-isopentenyl) adenosine (iPA) was quantified by an indirect ELISA using polyclonal antibodies, in the roots, xylem sap and leaves of pot grown sunflower plants subjected to water stress (RWC of leaves approximately 65 per cent). The delivery rates of all three cytokinins decreased significantly under stress. Cytokinin levels also decreased in roots and in leaves of stressed plants. Three-fold increase in cytokinin oxidase activity was observed in stressed roots after polymin P-ammonium sulphate fractionation. Further purification using Con A agarose resulted in elution of protein with cytokinin oxidase activity and was found to be 30 kDa protein on SDS-PAGE.