Direct Shoot Bud Formation and Tuberization from Aseptically Cultured Root Tubers of Calla Lily (Zantedeschia aethiopica L)

We describe here the development of a micropropagation protocol for mass multiplication of Zantedeschia aethiopica by using root tubers as explant. The surface sterilized root tubers produced five to six shoot-buds on semi-solid Murashige and Skoog’s (MS) medium with 10.0 mg l^?1 of 6-benzylaminopurine (BAP) and additives (50.0 mg l^?1 of ascorbic acid; 25.0 mg l^?1 each of adenine sulphate, L-arginine and citric acid). The cultures were multiplied by sub-culture of individual shoot bud produced in vitro and clumps of shoot buds generated in vitro in cultures on MS medium containing 3.0 mg l^?1 of BAP and additives. Further multiplication of propagules was achieved through tuber formation along with amplifying shoots on MS medium with 5.0 mg l^?1 of BAP. The micropropagated shoots were rooted both in vitro as well as ex vitro. Cent percent of the cloned shoots rooted in vitro within 15–18 days on hormone-free 1/2 strength MS salts with 200.0 mg l^?1 of activated charcoal. Alternatively 95–100% shoots rooted ex vitro under greenhouse conditions on soilrite after pulse-treatment with 500.0 mg l^?1 of Indole-3-butyric acid (IBA) or β-naphthoxyacetic acid (NOA) for 300 sec. The cloned plants were hardened in the greenhouse. The hardened plants were transplanted to soil for further acclimatization.     

Callus Culture and Plant Regeneration from Seedling Explants of Pergularia daemia (Forsk) Chiov

Callus cultures were established from seedling explants of Pergularia daemia (Forsk) Chiov on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins. Optimal callus developed from leaf explants on MS medium supplemented with 2,4-D (2 mg l^?1) + 2iP (0.1 mg l^?1), was used for morphogenesis. Adventitious shoots were regenerated (70%) from the calli on MS medium supplemented with NAA (0.1 mg l^?1)+ BAP (2 mg l^?1). Individual shoots were rooted on half strength MS medium supplemented with 0.1 mg l^?1 IBA. Plantlets with well developed roots were successfully transferred to soil and 50% of the transferred plants survived.     

In vitro Clonal Propagation and Organogenesis in Spilanthes acmella (L) Murray: A Herbal Pesticidal Plant of North-East India

Multiple shoots were regenerated in MS medium using different concentrations of BAP and Kn and different combinations of BAP with IAA, NAA and IBA. Highest multiplication of shoots was obtained with BAP (0.75 mg l^?1) with 28.4 shoots per explant after 60 days of culture. Shoots rooted best on IBA (0.5 mg l^?1), numbering 48.8 per explant. Organogenesis was maximum in callus cultured on MS medium supplemented with BAP (2.0 mg l^?1) and IAA (1.0 mg l^?1).     

In vitro Regeneration of Gerbera jamesonii Bolus from Leaf and Petiole Explants

Regeneration of adventitious shoots from leaf and petiole pieces of Gerbera jamesonii has been obtained on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins and cytokinins. About 75’77 per cent of the calli from both types of the explants produced 12’15 shoots per callus with 3 mg l^?1 SAP. Auxins and kinetin, separately failed to produce shoots. The shoots regenerated on the callus induction medium (elM). The regenerated shoots multiplied with 1 mg l^?1 SAP, were rooted on MS medium containing 1mg l^-1 BAP + 0.1 mg l^-1 IAA. The plants obtained were transferred to pots and acclimatized with 60’70 per cent success.     

In Vitro Clonal Propagation of Curcuma caesia Roxb and Curcuma zedoaria Rosc from Rhizome Bud Explants

Two species of Curcuma (C. caesia and C. zedoaria) have been propagated through tissue culture using rhizome bud explant. The best response for shoot multiplication was obtained on MS basal medium supplemented with 4 mg l^?1 BAP and 1.5 mg l^?1 NAA for C. caesia (3.5 ± 0.79 shoots per explant) and 1 mg l^?1 BAP + 0.5 mg l^?1 NAA for C. zedoaria (4.5 ± 0.15 shoots per explant). A maximum of 9.2 ± 0.15 and 8.9 ± 0.09 roots per explant were obtained for C. caesia and C. zedoaria, respectively when MS was supplemented with 0.5 mg l^?1 IAA. The rooted plants could be established in soil.     

Effect of Explant Source on Axillary Shoot Multiplication During Micropropagation of a Rare Medicinal Plant — Wattakaka volubilis (L f) Stapf

Nodal explants of in vivo plants and in vitro seedlings of Wattakaka volubilis were cultured on Murashige and Skoog medium fortified with various concentrations of cytokinins — BA (0.5–5 mg l^?1), KN (0.5–10 mg l^?1),TDZ (0.05–1 mg l^?1) either singly or in combination with NAA (0.1 mg l^?1). KN proved best for inducing healthy shoots in both in vitro and in vivo derived explants. Maximum number of shoots (14.1±0.84) with 80% regeneration frequency was obtained from nodal explants of seedlings cultured on 5 mg 1^?1 KN + 0.1 mg l^?1 NAA. In vivo nodal explants produced a maximum of 4.2 shoots on MS medium fortified with 2 mg l^?1 BA+0.1 mg l^?1 NAA. The differentiated shoots from both could be rooted with 85% frequency on 1/2 strength MS medium (1% sucrose) with 0.6% agar + 1 mg l^?1 IBA + 0.2 mg l^?1 KN. Rooted shoots were transplanted to vermiculite-soil (3:1) mixture in polyethylene covered pots with 45% transplantation success. Peroxidase isozymes (native PAGE) analysis helped to verify the variation in regenerated plants.     

Clonal propagation of Enset (Ensete superbum (Roxb.) Cheesman) through shoot tip cultures

Summary The morphogenetic potential of the shoot tip explants ofEnsete superbum (Roxb.) Cheesman, a wild relative of the cultivated bananas, was investigated and an effective clonal propagation method devised. Shoot tip explants grown in modified MS medium containing 1.5 mg l^–1 BAP and 1 mg l^–1 KIN developed corms which on transfer to medium containing 3 mg l^–1 IBA and 1.5 mg l^–1 BAP, regenerated a large number of shoots from the surface of the corm, the origin of which was traced to single hypodermal cells. Shoots were rooted on a half-strength MS medium salts containing 3 mg l^–1 IBA and 0.1 mg l^–1 BAP. The rooted plantlets were hardened and planted in the field where the plants looked normal.     

Micropropagation of Raisin Tree (Hovenia dulcis Thunb.) Through Axillary Bud Culture

Clonal propagation in vitro of raisin tree (Hovenia dulcis Thunb.) was achieved using axillary buds from mature trees and young plants. Explants cultured on Murashige-Skoog’s medium with 1/3 of the original salt concentration, supplemented with 0.5 mg l^-1 BAP and 0.5 mg l^-1 IAA, showed proliferation of new shoots in 4-5 weeks. Adventitious shoot proliferation was also stimulated in subsequent subcultures in the presence of BAP. The shoots rooted when transferred to 1/3 Murashlge and Skoog’s medium with 0.1 mg l^-1 of IBA. Plantlets thus formed were successfully transplanted to the field after a short acclimatization period.     

Micropropagation of Anethum graveolens L Through Axillary Shoot Proliferation

A protocol is described for rapid and large-scale in vitro propagation of Anethum graveolens by enhanced axillary shoot induction that was dependent on BAP supply. The synergistic combination of 0.5 mg l^?1 BAP and 0.1 mg l^?1 IBA induced 100% shoot formation as well as shoot number (6.6 ± 0.48 per explant). Subculturing of shoot tips of in vitro plants on multiplication medium enabled continuous production of healthy shoots with similar frequency. Rooting of shoots was achieved on a medium with 1mg l^?1 IBA and 0.5 mg l^?1 Kn. Micropropagated plants established in garden were uniform and identical to the donor plant with respect to morphological and cytological characteristics.     

High efficiency plant regeneration and genetic fidelity of regenerants by SCoT and ISSR markers in chickpea (Cicer arietinum L.)

High efficient and repeatable in vitro regeneration protocol was established from embryo axis, half-seed, axillary meristem, and cotyledonary node explants of chickpea. Various concentrations and combinations of various plant growth regulators (PGRs) were employed to induce multiple shoots, shoot elongation and rooting of shoots to obtain complete plantlets of chickpea. The pretreatment of seeds with 6-benzyl aminopurine (BAP) at 1.0 mg l^?1 was found to significantly increase the multiple shoot regeneration from the all explants tested. Among three PGRs such as BAP, kinetin (KIN) and thidiazuron (TDZ) tested for multiple shoot induction; BAP at 2.0 mg l^?1 produced the maximum number of shoots in all tested explants. The maximum number of shoots (48.80 shoots/explant) was attained from the embryo axis explant followed by half-seed (32.76 shoots/explant), axillary meristem (28.34 shoots/explant) and cotyledonary node explant (18.47 shoots/explant) on medium augmented with 2.0 mg l^?1 BAP along with 0.05 mg l^?1 Indole-3-butyric acid (IBA). The optimum percentage of shoot elongation response was recorded (96.68%) on medium fortified with IAA (0.05 mg l^?1), GA₃ (1.0 mg l^?1) and BAP (1.0 mg l^?1) with an average shoot length of 8.82 cm. The elongated shoots were successfully rooted in medium augmented with 2.0 mg l^?1 IBA. The complete plants were acclimatized in the greenhouse with a survival rate of 72%. The plantlets regenerated from four explants appeared to be morphologically similar to mother plants. The genetic fidelity of in vitro regenerated plants was evaluated using Start Codon Targeted and Inter simple sequence repeats molecular markers. The in vitro regenerated plants from all four explants were found to be the true to type with their mother plant. The in vitro protocol presented in the study should offer as a feasible system for chickpea genetic transformation.

     

Thidiazuron-induced Shoot Multiplication and Plant Regeneration in Bamboo (Dendrocalamus strictus Nees)

Thidiazuron (TDZ) stimulated shoot proliferation from different seedling explants (i.e., shoot, basal node, node and apical segment) of bamboo (Dendrocalamus strictus) when incorporated in half-strength Murashige and Skoog (MS) medium having 2% (w/v) sucrose. All the concentrations of TDZ (0.01 to 1.0 mg l^?1) tried were effective in shoot proliferation. Maximum shoots (14.8 ± 1.0) were obtained from the shoot explants cultured in 0.5 mg l^?1 TDZ supplemented halfstrength MS liquid medium for 21 days and subsequently transferred to the same medium devoid of TDZ. The longer culture period (i.e. 28 and 35 days) in TDZ medium caused reduction in shoot proliferation. The shoots regenerated with lower concentrations of TDZ treatment (i.e. 0.01 to 0.1 mg l^?1) rooted in half-strength MS liquid medium. The shoots formed with 0.5 mg l^?1 TDZ treatment did not root in basal medium and required auxin supplementation in the medium for rooting and about 55% shoots produced roots in 1.0 mg l^?1 IBA supplemented medium. The shoots formed with 1.0 mg l^?1 TDZ did not root even after auxin treatment. The well rooted shoots transplanted to plastic pots filled with sand and garden soil (1:1) mixture showed 98% establishment.     

Efficient Plant Regeneration System From Leaf Discs of Zonal (Pelargonium x hortorum) and two scented (P. capitatum and P. graveolens) Geraniums

An efficient and rapid plant regeneration system was established for zonal and scented geraniums using leaf discs as explants. Several explants, medium and culture conditions were studied to optimize shoot induction. Leaf discs taken from 4–5 weeks old in vitro grown plants, whatever the genotype, were more effective for shoot regeneration than those taken from greenhouse grown plants. Darkness proved to be a stimulating factor for shoot regeneration and the combination between NAA and two cytokinins gave the best results. Direct shoot regeneration (100%) was obtained from leaf discs of P. capitatum on half-strength MS medium supplemented with 0.5 mg l⁻¹ NAA in combination with 1 mg l⁻¹ of BAP and zeatin in darkness (11.4 shoots per explant). In the same medium and culture conditions, all P. graveolens leaf discs also exhibited direct shoot regeneration (7.3 shoots per explant). For P. x hortorum, 100% of leaf discs underwent shoot regeneration on a MS medium supplemented with 0.2 mg l⁻¹ NAA in combination with 0.5 mg l⁻¹ of BAP and zeatin in darkness (8.8 shoots per explant) or under low light conditions with 0.2 mg l⁻¹ NAA and 1 mg l⁻¹ of BAP and zeatin (7.5 shoots per explant). For this species, the best results for shoot elongation were obtained on half-strength MS medium gelled with Phytagel 0.3% (v/v). Whatever the genotype, all shoots rooted readily when transferred to diluted MS medium (MS/2) containing 1 mg l⁻¹ IAA. Acclimatized plants grew normally and flowered in greenhouse conditions. Flow cytometry analysis made on leaves of acclimatized plants revealed that all the scented geranium plants are similar to mother plants while 71% of P. x hortorum plants which showed strong growth were tetraploid.     

In Vitro Regeneration of Achillea millefolium L from Shoot-tips and Root Segments of Seedlings

This report describes, for the first time, an efficient plant regeneration system for Achillea millefolium L (yarrow), a medicinal plant, via shoot multiplication from shoot-tips and adventitious shoot regeneration from root segments. Higher numbers of shoots were obtained when shoot-tips were cultured on MSMO medium supplemented with 3.0 mg l^?1 BA and 0.5 mg l^?1 IAA, or 5.0 mg l^?1 KIN and 1.0 mg l^?1 IBA, producing 17.3 and 17.0 shoots per explant at 100% frequency, respectively. For adventitous shoot regeneration, only root segments developed shoots when cultured on medium containing a combination of 1 mg l^?1 TDZ, 0.5 mg l^?1 IAA and 0.5 mg l^?1 GA3 (18.9 shoots per explant at 100% frequency), while other types of explants (i.e., cotyledons, leaf lamina and petiole segments) or hormonal combinations tested were found ineffective. Regenerated shoots rooted readily on MSMO medium containing different concentrations of IAA, IBA, NAA or 2,4-D, however, NAA at 0.5 mg l^?1, or IBA at 0.5 or 1.0 mg l^?1 were found to be the most productive. Nearly all of the regenerated plants (98%) survived through the hardening process when the rooted plantlets were kept at 55–65% relative humidity for 2 weeks, which were then planted in pots containing potting soil and kept at 25–35% humidity.     

Embryogenesis,plant regeneration and cardiac glycoside determination in Digitalis ferruginea subsp. ferruginea L

The present study reports, for the first time, an efficient in vitro plant regeneration protocol for Digitalis ferruginea subsp. ferruginea L. (rusty foxglove). We have used different concentrations of gibberellic acid (GA₃) on Murashige and Skoog (MS) medium to assess the germination frequency of seeds. High frequency of germination was achieved on MS medium with 1.0 mg l^?1 GA₃. 6-Benzylaminopurine (BAP) combined with α-naphtaleneacetic acid (NAA) or 2, 4-dichlorophenoxy acetic acid (2, 4-D) in the induction MS medium induced both somatic embryogensis and shoot organogenesis. The highest percentage of callus growth (85 %) was obtained when hypocotyl explants were cultured on MS medium containing 0.5 mg l^?1 2, 4-D plus 1.0 mg l^?1 BAP. The maximum mean number of somatic embryos (7.3 ± 1.3 embryos) or shoots (12.0 ± 1.1 shoots) per callus was obtained when medium contained 0.25 mg l^?1 NAA plus 1.0 mg l^?1 BAP or 0.5 mg l^?1 NAA plus 2.0 mg l^?1 BAP. The regenerated shoots easily rooted on MS medium. Higher amounts of lanatoside C [13.2 ± 0.5 mg 100 g^?1 dry weight (dw)] and digoxin (2.93 ± 0.31 mg 100 g^?1 dw) accumulation were obtained when shoots were obtained by indirect regeneration. We also investigated derivatives of cardenolides, i.e., digitoxigenin (730 ± 180 mg 100 g^?1 dw), gitoxigenin (50 ± 20 mg 100 g^?1 dw) and digoxigenin (490 ± 170 mg 100 g^?1 dw) from natural samples.     

Rapid micropropagation of Woodfordia fruticosa (L.) Kurz (Lythraceae), a rare medicinal plant

A rapid propagation method comprising initiation of in vitro shoot tip culture from field-grown flowering plants and reculture of the nodal segments of regenerated shoots in Schenk and Hildebrandt (1972) medium was developed for Woodfordia fruticosa (L.) Kurz., a rare medicinal shrub. A medium supplement of 6-benzylaminopurine (0.2 mg.l^–1) induced high frequency (88%) development of axillary shoot buds (3.2) in 4–5 weeks. Subculture of the explants with multiple new shoots in fresh medium for 30 days yielded an even larger number (9.7) of shoots. Highest multiplication (26–35 shoots) was recorded when using culture initiation media with 0.5 mg.l^–1 each of BAP and NAA followed by subculture in 0.2 mg.l^–1 BAP. The shoot multiplication rate was further accelerated by reculturing 0.4–0.6 cm nodal segments of regenerated shoots in media with 1.0 mg.l^–1 BAP. Shoot cuttings (3.5–7.0 cm) were rooted in 0.2 mg.l^–1 IAA. Regenerated plants displayed uniform morphological, growth and flowering characteristics.Abbreviations BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - IAA Indole-3-acetic acid - IBA indole-3-butyric acid - SH Schenk and Hildebrandt (1972) medium     

In Vitro Method for Propagation of Centella asiatica (L) Urban by Shoot Tip Culture

A rapid clonal propagation system has been developed for the medicinally important herb Centella asiatica (L) Urban by shoot tip (2–3 cm long) culture. The shoot tips isolated from mature plants were inoculated on MS medium incorporated with BA alone or in combination with NAA and Kn. The optimum number of shoots (3.38) with optimum number of leaves per shoot (4.25) were attained on MS medium supplemented with 4.0 mg l^?1 BA and 0.1 mg l^?1 NAA. On transferring the microshoots on full strength MS medium supplemented with various concentrations of IBA (1.0-3.0 mg l^?1) and NAA (0.5-2.0 mg l^?1), profuse rooting (46.8 per shoot) was obtained in MS basal medium with 2.0 mg l^?1 IBA with root length of 19.7 cm. Well rooted plantlets were acclimatized successfully by adjusting the temperature and humidity for 3–4 weeks after transfer to pots filled with sterilized vermiculite soil: sand (1:1)mixture. This micropropgation protocol could be useful for raising a stock of genetically homogenous material for field cultivation within a very short period.     

In Vitro Plant Regeneration from Immature Leaflets Derived Callus of Acacia confusa Merr via Organogenesis

An efficient plant regeneration system was established from immature leaflet-derived callus of Acacia confusa Merr, through organogenesis. Under optimized culture conditions, the high rate of callus induction and proliferation was obtained in 35 days on MMS medium supplemented with 2,4-D (3 mg l^?1) + NAA (0.01 mg l^?1) + Kin (0.05 mg l^?1). The highest percentage of shoot regeneration response (95%) and greatest number of shoots (52.9) were obtained after the 46-day transfer of green nodular calli onto the shoot regeneration medium (WPM) supplemented with the BA 3 mg l^?1 + NAA 0.05 mg l^?1 + Zeatin 0.1 mg l^?1 + AdSO₄ 5 mg l^?1 combination. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoot buds to medium (half-strength MS) containing GA, (1 mg l^?1) and BA (0.05 mg l^?1), within 30 days. The elongated shoots were rooted on half-strength MS medium supplemented with 4 mg l^?1 IBA and 0.05 mg l^?1 Kin in the 42-day culture. Rooted plantlets were hardened and successfully established in soil. The field-established plants were morphologically normal and fertile.     

An efficient micropropagation protocol for Citrus jambhiri Lush. and assessment of clonal fidelity employing anatomical studies and RAPD markers

Citrus jambhiri (rough lemon) is considered a major rootstock source for a number of Citrus species. A simple method for micropropagation from nodal segments is reported. Nodal segments of C. jambhiri were inoculated on MS medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin, and N6-(2-isopentenyl) adenosine (2iP). Maximum multiple shoot regeneration response (75?%) was observed with BAP at 3?mg?l^?1. Shoots were multiplied for 30?d on fresh medium with similar composition. A total of 67?% of the cultures showed multiplication with the optimum number of shoots (4.02) and height of shoots (1.81?cm) with BAP (3?mg?l^?1) alone. Maximum rooting response (87?%) was observed with naphthaleneacetic acid at 0.5?mg?l^?1. Transverse sections of shoot stems obtained in vivo (sampled from seedlings) and in vitro (regenerated from nodal segments), showed similar anatomies. Randomly amplified polymorphic DNA analysis confirmed that all the regenerated plants were genetically identical to their donor plant, suggesting absence of detectable genetic variation in the regenerated plantlets.     

Regeneration of fertile Arachis paraguariensis plants from callus and suspension cultures

Highly morphogenic callus cultures were isolated from stamens of a wild peanut species, Arachis paraguariensis. These cultures were initiated on modified N6 medium containing 0.2 mg1l^-1 4amino-3,5,6-trichloro-picolinic acid (picloram) and 0.5 mg l^-1 6-benzylaminopurine (BAP) and were maintained on modified N6 medium with 0.008 mg l^-1 picloram and 0.25 mg l^-1 BAP. Buds formed on the calli growing on the maintenance medium developed into shoots when they were transferred to a MS salts based medium with no hormones. The cultures could also be maintained as a suspension culture in N6 liquid medium. When cell clumps larger tham 840 m were collected from the suspension culture and transferred to MS medium without hormones, they formed shoots in liquid culture. Root formation rarely occurred in agar or liquid cultures. Therefore, grafting to stems of rooted seedlings was used to obtain plants from regenerated shoots. Eight out of 50 field grown plants produced viable seed.     

Regeneration of Heracleum candicans Wall Plants from Callus Cultures through Organogenesis

Callus was initiated from petiole explants of Heracleum candicans on MS medium fortified with BAP and 2,4-D ( 0.5 mg I^-1 each). Maximum shoot differentiation from callus occurred on MS medium containing 1 mg I^-1 BAP and 0.2 mg I^-1 NAA. The regenerated shoots were rooted on MS medium supplemented with 1 mg I^-1 IBA. The rooted plants were transferred to the field after successful hardening in pots containing vermiculite. All regenerated plants were diploid with 2n=22 chromosomes in their root tip cells.