Single-node Explants Research Articles

Conservation of Colutea gifana, a rare and potential ornamental species, using in vitro method

In vitro methods provide a variety of tools to supplement traditional methods for collection, propagation and preservation of endangered plant species. In this study, an efficient protocol was developed for in vitro propagation of Colutea gifana, a rare and endangered plant species with limited reproductive capacity that grows in a narrow area of Iran. Single node explants were used for a series of experiments to select the appropriate disinfection method and growth regulators for establishment, proliferation and rooting stages. Explants showed the highest establishment percent after treatment with 2% Sodium hypochlorite (NaOCl) for 15 min, cultured in MS medium containing 2.2 μM 6-benzylaminopurine (BA) and 1 μM indole-3-butyric acid (IBA). In proliferation stage, 8.8 μM of BA was more effective cytokinin than Kinetin (Kin) and Thidiazuron (TDZ) for growth induction of axillary shoots. In vitro rooting of proliferated shoots was induced in halfstrength MS medium in all concentrations of both tested auxins i.e. IBA and α-naphthalene acetic acid (NAA). Eighty percent of the plantlets were successfully acclimatized to ex vitro conditions, showed normal development. These plants were used to replenish declining populations in the collection sites and conserve C. gifana from extinction.

Effect of in vitro topophysis on the growth, development, and rooting of chrysanthemum explants (Chrysanthemum grandiflorum /Ramat./ Kitam)

SummaryThe consequences of using ex vitro, single-node explants from different topophysical positions in chrysanthemum (Chrysanthemum grandiflorum /Ramat./ Kitam) were determined. In particular, how explant topophysis affected the rate of propagation, which is important for the successful micropropagation of chrysanthemum. Uniform shoots of five cultivars of chrysanthemum, cultured in vitro, were each divided into three equal zones: distal, central, and proximal. Two single-node explants were isolated from each zone and cultured on MS medium without any added growth regulators. After 10 weeks of culture, 50% of the shoots that had developed from axillary buds on each single-node explant were excised and measurements were taken in order to compare those shoots that had developed from explants from the different topophysical zones. The remaining shoots were sub-cultured on rooting medium. After 4 weeks, the numbers of roots per plantlet, and the total fresh weight (FW) of roots were recorded. The cultivars fell into two groups. ‘Lady Amber’, ‘Lady Orange’, and ‘Lady Vitroflora’ explants were topophysis-dependent, while ‘Lady Bronze’ and ‘Lady Rosy’ explants were topophysis-independent. For the three topophysis-dependent cultivars, the propagation rate, growth rate, shoot length, internode length, single leaf weight, and total plantlet FW values were highest for those shoots derived from the central and proximal zones. Topophysis failed to affect the number of leaves per shoot or the number of days between the appearance of two successive leaves. The effects of topophysis on the number of roots per plantlet and on root FW were inconsistent. The unequal growth of chrysanthemum plantlets during in vitro micropropagation can be an effect of topophysis, and this phenomenon is cultivar-specific in chrysanthemum.

Micropropagation of Ocimum kilimandscharicum Guerke (Labiatae)

An efficient plant regeneration protocol has been developed from nodal explants of Ocimum kilimandscharicum Guerke, a medicinally important herbaceous plant species belonging to the family Lamiaceae. Axillary shoot bud proliferation was initiated from nodal explants cultured on MS medium supplemented with various concentrations of 6-benzyladenine (BA) (0.5–3.0 mg/l), kinetin (KN) (0.5–3.0 mg/l) and 2-isoPentenyladenine (2-iP) (0.5–3.0 mg/l). The maximum number of shoots (6.09±0.05), with average length 3.83±0.11 cm, was achieved with medium containing 1.0 mg/l BA. Shoot culture was established by repeated subculturing of the original nodal explants on shoot multiplication medium after each harvest of newly formed shoots. In this way, 20–30 shoots were obtained from a single nodal explant after 5 months. Rooting of shoots was achieved on half-strength MS medium supplemented with 1.5 mg/1 Indole-3-butyric acid (IBA) and 2% sucrose. Well-developed plantlets transferred to plastic pots containing soil and vermiculite (1:1) showed 81.13% survival. The genetic fidelity of in-vitro-raised field-grown plants to the donor plant was ascertained from random amplified polymorphic DNA (RAPD) markers. This protocol can be used for commercial propagation and for future genetic improvement studies.

Micropropagation and bioreactor studies of the medicinally important plant Lessertia (Sutherlandia) frutescens L.

This study describes a protocol for rapid and efficient in vitro propagation of Lessertia frutescens (cancer bush), a medicinally important plant species native to southern Africa. Single node explants were grown in various culture regimes of MS medium containing 30g/l sucrose supplemented with various concentrations of cytokinins and auxins and solidified with 8g/l agar. These were (a) 2.22, 4.44, 13.32 and 22.19µM BA; 2.32, 4.65, 13.95 and 23.23µMK and 0.45, 2.27, 4.54 and 13.62µM TDZ (b) a combination of 2.22µM BA with 0.57, 2.85, 5.71 and 11.42µM IAA, 0.49, 2.46, 4.9 and 9.8µM IBA or 0.54, 2.69, 5.37 and 10.74µM NAA and (c) different media types viz. MS, SH basal salt medium and WPM at 1, ½ and ¼ salt strength which were each supplemented with 2.22µM BA and 0.54µM NAA. Single node explants were also grown in MS liquid medium supplemented with 2.22µM BA and 0.54µM NAA in temporary and continuous immersion bioreactors. Maximum number of shoots (12.9) per single node explant was obtained in the temporary immersion bioreactor but 50% of these shoots showed symptoms of hyperhydricity. In solid culture the best shoot multiplication response (10 shoots) was obtained in full strength MS. Roots were induced using shoot tips cultured in ½ MS solid medium supplemented with various concentrations of IBA or NAA. The highest rooting percentage (78%) was achieved in 19.6µM IBA. Rooted plantlets were cultured in a mixture of perlite and vermiculite (1:1; v/v) and successfully acclimatized in a growth chamber with an 85% survival rate.

Cryoconservation and regeneration of axillary shoot meristems of Indigofera tinctoria (L.) by encapsulation–dehydration technique

In vitro axillary shoot proliferation was achieved from single-node explants of Indigofera tinctoria on a well-defined medium, Murashige and Skoog (MS) medium supplemented with 1.0 mg l−1 N 6-benzyl adenine (BA) and 0.1 mg l−1 indole-3-acetic acid. Axillary shoot meristems from cultures derived from up to three subcultures were used in the encapsulation–dehydration technique. Preconditioned, calcium alginate-encapsulated, and precultured axillary shoot meristems were subjected to different lengths of desiccation in a laminar flow cabinet. Maximum survival and regeneration rates of 56.7% and 62.2%, respectively, were obtained in half-strength (half the macro- and micronutrients and full-strength vitamins) MS medium supplemented with 0.5 mg l−1 gibberellic acid and 0.2 mg l−1 BA after 4 h of desiccation, during which the moisture content was reduced to 16.0%. According to the analysis of six random amplified polymorphic DNA markers, plantlets derived from cultures initiated with cryopreserved plant material were genetically identical to those derived from nonfrozen (control) tissues.

Direct regeneration and in vitro flowering of Scoparia dulcis L

This study reports a simple micropropagation protocol and thereby rapid multiplication of the useful medicinal plant- Scoparia dulcis L. Single node explants were inoculated on basal MS medium containing 3% (w/v) sucrose, supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin (KN), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and Naphthalene acetic acid (NAA) for direct plant regeneration. Maximum numbers of shoot (~22) were observed on the medium containing 0.5 mg/l BAP and 0.25 mg/l IAA after four weeks of culture. Regenerated shoots were separated and rooted on half strength MS medium supplemented with 0.5 mg/l of IBA alone for three weeks. Simultaneous regeneration of shoots and roots and in vitro flowering were achieved from the nodal explants on MS medium supplemented with 0.5 mg/l KN and 2.0 mg/l IAA. Well-developed complete plantlets were transferred on to specially made plastic cup containing soil rite. Acclimatized plantlets were successfully grown in garden soil.

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

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.

English

Single nodal explants excised from vines of greenhouse-grown white Guinea yam (Dioscorea rotundata poir cv Obiaoturugo) were initiated in vitro on a medium consisting of Murashige and Skoog’s (MS) basal salt supplemented with Gamborg’s B5 vitamins, 0.5 µM benzylamino purine (BAP), 0.1 µM naphthalene acetic acid (NAA), 0.2 µM gibberellic acid (GA3), 20 mgl-1 L-cysteine, 30 gl-1 sucrose and solidified in 8 gl-1 agar. They were incubated for 14 months with regular monthly subculture under continuous illumination at 24 ± 1oC and 1000 lux light intensity. Subsequently, single and double leafed segments from these were transferred to a simpler liquid medium made up of MS basal salts and vitamins, reduced sucrose level (20 gl-1) and without growth regulators. The cultures were kept stationary, and without subculture for 6 months at 27 ± 1oC, 16 h illumination and at 2000 – 2500 lux light intensity. The shoot cultures began to produce excessive roots at the nodes apart from the shoot tip. Subsequently microtubers developed at the position of the axiliary buds subtended by the leaf petiole as well as at the base of some shoots. On transfer whole or segmented into fresh medium, the microtubers sprouted and produced plantlets. Key words: Dioscorea rotundata, nodal, microtuberization, nutritional stress.

In Vitro Clonal Propagation and Medium Term Conservation of Brahmi [Bacopa monnieri (L) Wettst

A protocol has been developed for in vitro clonal propagation leading to conservation of Bacopa monnieri (L) Wettst, a medicinal plant of high commercial potential with legendary reputation as a memory vitalizer. Single node explants when cultured on Murashige and Skoog’s medium supplemented with BA (0.2 mg l-1), showed active shoot proliferation (22.2 shoots/ explant in 8 weeks) without callus formation. Rooting was achieved on the same medium. The generated shoots could also be conserved for 12 months with high survival rate (up to 100%). The regenerants upon transfer to soil showed no morphological variation as compared with the donor plants. The medium optimized in the present study was applied for culture establishment and conservation of a total of 15 Brahmi accessions procured from different regions.

In vitro basal and nodal microtuberization in yam shoot cultures ( Discorea rotundata poir, cv. Obiaoturugo) under nutritional stress conditions

Single nodal explants excised from vines of greenhouse-grown white Guinea yam (Dioscorea rotundata poir cv Obiaoturugo) were initiated in vitro on a medium consisting of Murashige and Skoog’s (MS) basal salt supplemented with Gamborg’s B5 vitamins, 0.5 iM benzylamino purine (BAP), 0.1 iM naphthalene acetic acid (NAA), 0.2 iM gibberellic acid (GA3), 20 mgl-1 L-cysteine, 30 gl-1 sucrose and solidified in 8 gl-1 agar. They were incubated for 14 months with regular monthly subculture under continuous illumination at 24 ± 1oC and 1000 lux light intensity. Subsequently, single and double leafed segments from these were transferred to a simpler liquid medium made up of MS basal salts and vitamins, reduced sucrose level (20 gl-1) and without growth regulators. The cultures were kept stationary, and without subculture for 6 months at 27 ± 1oC, 16 h illumination and at 2000 – 2500 lux light intensity. The shoot cultures began to produce excessive roots at the nodes apart from the shoot tip. Subsequently microtubers developed at the position of the axiliary buds subtended by the leaf petiole as well as at the base of some shoots. On transfer whole or segmented into fresh medium, the microtubers sprouted and produced plantlets.

THE INFLUENCE OF LIGHT COLOUR ON MICROPROPAGATION OF CHRYSANTHEMUM

Single-node explants of chrysanthemum (Dendranthema grandiflora Tzvelev) 'Lilac Wonder' were cultured in vitro during 6 weeks on MS medium without growth regulators under different light: red, yellow, green, blue, white. The influence of light colour on growth and regeneration of plantlets originated from axillary bud was investigated. Green light showed the highest positive influence on stems length and fresh weight, length of internodes and propagation efficiency of plantlets. Plantlets cultured under blue light had the shortest internodes and stems, though their weight was not the lowest, which resulted in thickset shape of plantlets and the worst propagation efficiency. Microcuttings were well rooted under both green and white light. Plantlets grown under red light had the least weight, tiny leaves and were predominantly unrooted. Light colour showed no significant influence on an average number of leaves per plantlet. No branching was observed under any light conditions. Using green light may be helpful in improving the efficiency of micro-, propagation of chrysanthemum.

An efficient, in vitro cyclic production of shoots from adult trees of Crataeva nurvala Buch. Ham.

An efficient, cyclic, two-step protocol for micropropagation of medicinal tree, Crataeva nurvala has been successfully developed, which can be employed at a commercial scale. Nodal explants from 30-year-old tree when cultured on MS medium supplemented with 2.22 microM BAP produced multiple shoots, which elongated satisfactorily on the same medium. Nodal and leaf explants from in vitro regenerated microshoots too developed shoots, thus making the process recurrent. In 6-month duration, owing to the recurring nature of the protocol, over 5400 shoots could be produced from a single nodal explant from the adult tree. Addition of casein hydrolysate significantly increased the average number of shoots per explant. Maximum number of shoots regenerated on medium supplemented with 100 mg l(-1) casein hydrolysate. Shoots could be rooted on 1/2 MS supplemented with 0.11 and 0.54 microM NAA. Regenerated plantlets were acclimatized and successfully transplanted to soil.

Micropropagation and encapsulation of medicinally important Chonemorpha grandiflora

Abstract This study describes a protocol for in vitro propagation of Chonemorpha grandiflora (Roth) S.M. & M.R. Almeida (Apocynaceae) through axillary bud proliferation and rooting. For shoot induction, the combination of 13.3 μM N6-benzyladenine (BA) and 2.46 μM indole-3-butyric acid (IBA) in Murashige and Skoog (MS) medium was better than other growth regulators. Liquid medium was superior over solid medium for root formation and growth. IBA was more effective than other auxins for root induction. Addition of silver nitrate in the presence of IBA significantly increased the number, length, and branching of roots. Shoot tips encapsulated in medium containing 0.49 μM IBA and 11.7 μM silver nitrate exhibited 95% conversion to plantlets. This protocol enables the production of 250 plantlets from a single nodal explant within 7 mo. Plantlets showed 90% survival after acclimatization in soil and were morphologically similar to the source plant under field conditions.

Changes of free, soluble conjugated and bound polyamine titers of jojoba explants under sodium chloride salinity in vitro

Jojoba (Simmondsia chinensis L.) single node explants were cultured in a basal medium supplemented with 17.8 microM 6-benzyladenine and four levels of sodium chloride concentration (0, 56.41, 112.82 and 169.23 mM). The free, the soluble conjugated and the insoluble bound forms of polyamines (PAs) (putrescine (Put), spermidine (Spd) and spermine (Spm)) were determined monthly during a 3-month proliferation stage. Free Put and Spd were found in higher levels in the control treatment, while Spm content was higher in the salt treatments. All soluble conjugated PAs were found to be in lower concentrations in explants growing on medium supplemented with salt, while the opposite was true for the insoluble bound PAs. It appeared that certain PAs and PAs forms could play a significant role in the adaptation mechanism of jojoba under saline conditions.

In vitro multiplication of Ocimum gratissimum L. through direct regeneration

The objective of this study was to develop a rapid system for regeneration of the important medicinal plant, Ocimum gratissimum L, from nodal explant. Single node explants were inoculated on basal MS (Murashige and Skoog, 1962) medium containing 3% (w/v) sucrose, supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin (KN), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA) for direct plant regeneration. Maximum numbers of shoot (14.3± ± ± ±1.5) were observed on the medium containing 0.5 mg/l BAP and 0.25 mg/l IAA after four weeks of culture. Regenerated shoots were separated and rooted on same half strength MS medium supplemented with 0.5 mg/l of IAA alone for three weeks. Well-developed complete plantlets were transferred on to specially made plastic cup containing soilrite. Acclimatized plantlets were successfully grown in garden soil.

An efficient method for explant sterilization for reduced contamination

An improved method for sterilization of explants was tested and found to be suitable for plants with elongated internodes, such as bamboos. Final cutting of the explants into single nodal segments for inoculation was done only after surface sterilization of multi-nodal explants in a stoppered glass measuring cylinder. This minimized penetration of the contaminants and the sterilizing agents into the exposed intercellular spaces and vascular cavities at the cut ends, thereby minimizing their harmful effects. The method was experimented upon three different plants, viz., bamboo, tea and rose. Through this method the number of cultures getting contaminated was substantially reduced as compared to the conventional means where single nodal explants were used, employing identical treatments. Moreover, in this method, the number of cultures showing bud-break also showed a marked increase thereby resulting in a tremendous increase in the percentage of successfully established proliferating cultures.

In vitro micropropagation of endangered Rhododendron ponticum L. subsp. baeticum (Boissier & Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Anderson’s modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months).

Factors affecting tissue culture of Damask rose ( Rosa damascena Mill.)

The present study was conducted to evaluate the regeneration ability of Damask rose. Single-node explants were surface sterilised with 10% chlorox for 15 min and cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N 6-benzyladenine (BA) or kinetin (Kin) separately or in combination with different concentrations of indole-3-butyric acid (IBA). Combination of 2.5–3 mg/l BA and 0.1 mg/l IBA was the most suitable treatment for proliferation. In vitro derived shoots were subcultured four times on the fresh medium within a 4-week period. Other treatments such as explant orientation (horizontal, vertical and oblique) and explant wounding were also examined but did not affect shoot multiplication rate significantly. Several experiments were carried out to stimulate in vitro rooting of Damask rose. Application of different media such as MS, 1/2 MS, 1/3 MS and 1/4 MS with different concentrations of indole-3-acetic acid (IAA), IBA and naphthaleneacetic acid (NAA) did not produce satisfactory results. Quick-dip method using sterilised 0–2000 mg/l IAA, IBA and NAA solutions was also studied. Other treatments such as using various concentrations of abscisic acid (ABA) in combination with various concentrations of IAA, IBA and NAA, and using various concentrations of sucrose and agar did not produce any roots. The best treatment for rooting of shoots was 2.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 2 weeks in MS medium and then transferring the explants to MS medium without any growth regulator. Plantlets were acclimatised in a soil mixture consisting of peat moss and sand 1:1 (v/v) and successfully transferred to the greenhouse after 3 weeks.

Carboxylic acids affect induction, development and quality of potato (Solanum tuberosum L.) microtubers grown in vitro from single-node explants

The role of three carboxylic acids with increasing alkyl-chain length, viz., formic, acetic and propionic acids in microtuberization was investigated in three potato (Solanum tuberosum L.) genotypes in vitro. Different concentrations of these carboxylic acids (0.0, 1.5, 3.0, 4.5 and 6.0 mM) were supplemented in microtuber induction medium, which was based on MS medium containing 8% sucrose, and their efficacy for induction, development and quality of microtubers was studied using single-node explants under continuous darkness at 20 °C. The carboxylic acids exhibited a strong stolon- and root-inhibiting effect on single-node explants with their increasing concentrations as well as alkyl-chain length (i.e., formic < acetic < propionic acids), and their mode of action was synonymous with antigibberellin substances. However, they did not have any significant inductive effect on microtuberization as compared to that under 8% sucrose medium. Rather they did show a detrimental effect on microtuber development in terms of average microtuber fresh weight with increasing concentrations as well as alkyl-chain length; both acetic and propionic acids at 6.0 mM induced the smallest microtubers in vitro. The carboxylic acids could, however, significantly increase the harvest indices suggesting their possible role in the regulation of source-sink co-ordination during microtuberization from single-node explants. But the most favourable effect of carboxylic acids on microtubers was apparent in terms of dry matter concomitant with higher starch synthesis and enhanced accumulation of reducing and total sugars. Acetic acid was the most effective in increasing the percentage of microtuber dry matter. The higher percentage of dry matter with higher carbohydrate reserves in microtubers induced by the carboxylic acids could be assumed to affect the quality of microtubers for subsequent storage, dormancy release and sprout growth.

Effect of leaves on microtubers produced from potato single-node cuttingsIn Vitro

Microtubers are used to propagate, to store, and to transport potato clones. Culturing single-node explants from potato plantletsin vitro without subtending leaves was reported to result in plantlets with lower vigor and a higher coefficient of variation. The effect on microtuber productionin vitro of leaf area and the presence or absence of leaves on potato single-node cuttings was investigated as an extension of the above study. Stock plantlets of potato cvs Atlantic, Kennebec, Russet Burbank, and Shepody were cultured under a 16-h photoperiod. Single-node cuttings were excised and grown in a high-sucrose tuberization medium in darkness. Leaf area did not affect the frequency, size, or weight of microtubers of cvs Katahdin and Russet Burbank. The absence of leaves reduced microtuber diameter for Russet Burbank; whereas Atlantic, Kennebec, and Shepody were unaffected. Mean fresh weight of microtubers was reduced when leaves were removed for all cvs except Atlantic. No effect of the removal of the leaf was observed for mean dry weights of microtubers from all cvs, although microtubers from single-node cuttings without leaves accumulated significantly more percent dry matter than those with leaves. Rapid multiplication facilities may therefore wish to consider conserving resources such as media, vessels, and growth room space by culturing explants without leaves for the production of microtubers.