Callus mediated shoot organogenesis and regeneration of cytologically stable plants of Ledebouria revoluta: An ethnomedicinal plant with promising antimicrobial potency

Ledebouria revoluta are important ethnomedicinal plant found in India and South Africa. Micropropagation via indirect shoot organogenesis had been established from three types of explant (i.e. scale leaf, leaf lamina and root) of L. revoluta. Scale leaf was found superior as compared to leaf lamina and root explant with respect to their organogenic callus induction potentiality. Murashige and Skoog (1962) [MS] media supplemented with 3.0?mg?L^?1 2,4-dichlorophenoxyacetic acid, 0.75?mg?L^?1 β-naphthoxyacetic acid were best effective for inducing organogenic callus. Maximum 17.0?±?0.52 bulblets were induced from about 500?mg of callus within 42–46?days sub-culturing on a medium containing 0.75?mg?L^?1 kinetin. The bulblets were matured (86.7% success) after one month culture on the same medium composition. The best result of in vitro root induction with 100% response and 8.4?±?0.31 roots per bulb was achieved after 18?days of implantation on MS medium containing 2.0?mg?L^?1 indole-3-butyric acid. Plantlets were acclimatized with a 96.0% survival rate. Chromosomal studies revealed cytological stability of callus cells and all regenerants containing 2n?=?30 chromosomes, same as parental plants. Antimicrobial activity of L. revoluta was tested against two Gram-positive bacteria, three Gram-negative bacteria and two fungi. The methanol and ethanol extract proved more effective against bacteria, whereas acetone and chloroform extract shows potential anti-fungal activities. Present protocol can be applied reliably to produce uniform planting materials in large scale. In addition, this efficient indirect regeneration pathway via callus culture opens a way for improvement through genetic transformation.     

Plant regeneration from phyllode explants of Acacia crassicarpa via organogenesis

In this paper we report the establishment of Acacia crassicarpa regeneration through organogenesis. We used phyllode (leaf) explants excised from 60-day-old in vitro seedlings for green compact nodule induction and, tested Murashige and Skoog (MS) media supplemented with various concentrations of 1-phenyl-3-(thiadiazol-5-yl) urea (thidiazuron) (TDZ) and α-naphthaleneacetic acid (NAA). Under the optimized condition, green compact nodules and adventitious shoots were induced in 10 and 40 days, respectively, on the medium containing a combination of 0.5 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA. This medium also yielded the highest rate (56%) of adventitious shoots forming from the nodules. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoots to medium containing 0.1 mg l⁻¹ TDZ within 2 months. The elongated adventitious shoots were rooted at a rate of 96.5% on half-strength MS medium with 0.5 mg l⁻¹ 3-indolebutyric acid (IBA) in 1 month. Rooted plantlets were hardened and successfully established in soil with an 80% survival rate. To our knowledge, this is the first report describing a detailed protocol for regeneration through organogenesis using phyllodes as explants for A. crassicarpa.     

Plant regeneration from <Emphasis Type="Italic">Gossypium davidsonii</Emphasis> protoplasts <Emphasis Type="Italic">via</Emphasis> somatic embryogenesis

Protoplasts isolated from wild cotton Gossypium davidsonii were cultured in KM₈P medium supplemented with different phytohormones. The most effective combination was 0.45 μM 2,4-dichlorophenoxyacetic acid, 2.68 μM α-naphthaleneacetic acid and 0.93 μM kinetin and the division percentage at the 8^th day was 30.78 ± 3.04 %. The density of protoplasts at 2–10 × 10⁵ cm⁻³ was suitable for protoplast division and calli formation, with a division percentage of 32.21 ± 3.64 % and a plating efficiency of 9.12 ± 2.61 % at the 40^th day. The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol. Protoplasts were cultured in three ways, a double-layer culture system, with liquid over solid medium was proved to be the best way. Embryo induction was further increased by addition of 0.14 μM gibberellic acid.     

In vitro shoot and root organogenesis, plant regeneration and production of tropane alkaloids in some species of Schizanthus

A rapid in vitro propagation system leading to formation of shoots from callus, roots, and plantlets was developed for Schizanthus hookeri Gill. (Solanaceae), an endemic Chilean plant. The genus Schizanthus is of particular interest due to the presence of several tropane alkaloids. So far, in vitro propagation of species of this genus has not been reported. Propagation of S. hookeri consisted of two phases, the first one for callus initiation and shoot formation and the second for rhizogenesis and plantlet regeneration. From a single callus that rapidly increased in cell biomass (from approximately 50 mg to approximately 460 mg/culture tube [25 x 130 mm] in 60 days) in the presence of 2.69 microM NAA and 2.22 microM BA, more than 10 shoots/callus explant were formed. From the latter, approx. twenty plantlets formed after 90-110 days shoot subculture in medium devoid of growth regulators that favored root formation. Ten alkaloids ranging from simple pyrrolidine derivatives to tropane esters derived from angelic, tiglic, senecioic or methylmesaconic acids were obtained from in vitro regenerated plantlets. One of them, 3alpha-methylmesaconyloxytropane, was not previously described. The same growth conditions, as well as other growth regulator levels tested, were required to induce callus and root formation in S. grahamii Gill. Root organogenesis occurred despite a high level of BA vs. NAA used, (i.e., 4.44 microM BA and 0.54 microM NAA); however no shoot formation was achieved. In the case of S. tricolor Grau et Gronbach, only callus formation was obtained in the presence of various growth regulators.     

Direct somatic embryogenesis from leaves,cotyledons and hypocotyls of <Emphasis Type="Italic">Hippophae rhamnoides</Emphasis>

Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in seabuckthorn (Hippophae rhamnoides L.) was achieved. The influences of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants were studied. The highest frequency of somatic embryos production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg dm⁻³ kinetin and 0.2, 0.5 mg dm⁻³ indole-3-acetic acid. Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55 %. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.     

Induction of synchronous secondary somatic embryogenesis in Camellia sinensis (L.) O. Kuntze

Nutritional effect of nitrate salts of potassium and ammonium, together with different concentrations of sulphate salts of aluminium, potassium, magnesium, and ammonium on secondary somatic embryogenesis, wereinvestigated. Nitrate salts of potassium (9.39 mmol/L) and ammonium (10.31 mmol/L) with only 1.5 mmol/L potassium sulphate produced maximum number of synchronous secondary embryos (i.e. 20-25 secondary embryos per primary embryo in 91.6 percnt; responsive explants).Of the different factorial combinations of glutamine, BAP, and IBA tested, maximum number of synchronous secondary embryos developed on a medium supplemented with 8.88 μmol/L BAP, 0.98 μmol/L IBA and 10 mmol/L glutamine.Synchronous and normal development of secondary embryos could thus be obtained when optimal concentrations of PGRs, glutamine, nitrates, and salts of potassium sulphate were combined together.Germination of the embryos (up to 52 percnt;) was acheived only when sulphate salts of potassium were removed from the medium.     

Somatic embryogenesis, scanning electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six date palm (Phoenix dactylifera L.) cultivars

An efficient somatic embryogenesis system has been established in six date palm (Phoenix dactylifera L.) cultivars (Barhee, Zardai, Khalasah, Muzati, Shishi and Zart). Somatic embryogenesis (SE) was growth regulators and cultivars dependent. Friable embryogenic callus was induced from excised shoot tips on MS medium supplemented with various auxins particularly 2,4-dichlorophenoxyacetic acid (2,4-D, 1.5 mg 1^−l). Suspension culture increased embryogenesis potentiality. Only a-naphthaleneacetic acid (NAA, 0.5 mg 1⁻¹) produced somatic embryos in culture. Somatic embryos germinated and converted into plantlets in N⁶-benzyladenine (BAP, 0.75 mg 1^−l) added medium following a treatment with thidiazuron (TDZ, 1.0 mg 1^−l) for maturation. Scanning electron microscopy showed early stages of somatic embryo particularly, globular types, and was in masses. Different developing stages of embryogenesis (heart, torpedo and cotyledonary) were observed under histological preparation of embryogenic callus. Biochemical screening at various stages of somatic embryogenesis (embryogenic callus, somatic embryos, matured, germinated embryos and converted plantlets) of date palm cultivars has been conducted and discussed in detail. The result discussed in this paper indicates that somatic embryos were produced in numbers and converted plantlets can be used as a good source of alternative propagation. Genetic modification to the embryo precursor cell may improve the fruit quality and yield further.     

Micropropagation of apple — A review

Micropropagation of apple has played an important role in the production of healthy, disease-free plants and in the rapid multiplication of scions and rootstocks with desirable traits. During the last few decades, in apple, many reliable methods have been developed for both rootstocks and scions from a practical, commercial point of view. Successful micropropagation of apple using pre-existing meristems (culture of apical buds or nodal segments) is influenced by several internal and external factors including ex vitro (e.g. genotype and physiological state) and in vitro conditions (e.g., media constituents and light). Specific requirements during stages of micropropagation, such as the establishment of in vitro cultures, shoot multiplication, rooting of microshoots and acclimatization are summarized in this review. New approaches for increasing shoot multiplication and rooting for apple and current use of micropropagated plantlets as tools in basic and applied research are also discussed.     

In vitro biotechnological advancements in Malabar nut (Adhatoda vasica Nees): Achievements,status and prospects

Adhatoda vasica Nees, belonging to family Acanthaceae, is a well-known medicinal plant. It is endorsed for its pyrroloquinazoline alkaloids and its derivatives, such as vasicine and vasicinone. Germinating A. vasica seeds is a tedious task; on that account, vegetative propagation is the preferred method for its multiplication. For rapid and large-scale multiplication, germplasm conservation as well as secondary metabolites production, in vitro culture of A. vasica was preferred over conventional propagation by several researchers; however, some major applications of this tissue culture technique are still awaiting to undergo extensive research. The present review, for the first time, illustrates all the major achievements associated with in vitro regeneration of A. vasica, reported till date and highlights the future prospects.     

CUC2 as an early marker for regeneration competence in Arabidopsis root explants

CUP SHAPED COTELYDON 2 (CUC2) was tested as a marker for shoot induction to monitor and facilitate the optimization of in vitro regeneration of Arabidopsis thaliana. The expression of a pCUC2::3XVENUS-N7 fluorescent marker allowed the observation of early steps in the initiation and development of shoots on root explants. The explants were first incubated on an auxin-rich callus induction medium (CIM) and then transferred to a cytokinin-rich shoot induction medium (SIM). CUC2-expression occurred prior to visible shoot formation during the incubation of the root explant on CIM. Shoot formation was invariably preceded by the accumulation of CUC2 expression at dispersed sites along the root explant. These patches of CUC2-expression also marked the site of lateral root primordium formation in root explants that were transferred to hormone free medium. Thus, CUC2 is a predictive marker for the acquisition of root explant competence for root and shoot organogenesis.