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.     

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.     

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.     

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.     

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.     

In Vitro Propagation of Endangered Temperate Himalayan Medicinal Herb Picrorhiza kurroa Royle ex Benth using Leaf Explants and Nodal Segments

The present study describes the micropropagation of Picrorhiza kurroa, (commonly known as kutki) an endangered medicinal herb of the temperate Himalayas and a source of hepatoprotective picrosides. In vitro shoot multiplication was achieved through sprouting of axillary buds using nodal segments and leaf tissue. For shoot regeneration, the hormone combinations kinetin (2.0 mg l^?1) and Kinetin + Indole-3-butyric acid (IBA) (2.0 mg l^?1 + 0.50 mg l^?1) with leaf explant was found superior. Interestingly, the basal MS medium gave 99.94 % response (direct proliferation) with nodal explant. The medium supplemented with IBA (1.0 mg ^?1) was found best for rooting of regenerated shoots. Nodal segments plated on the medium supplemented with TDZ + IBA (0.11 mg ^?1 + 0.50 mg ^?1) formed somatic embryos, however further regeneration could not be achieved. The in vitro raised plantlets were hardened and successfully established in the glass house conditions.     

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.     

Micropropagation of Withania coagulans (Stocks) Dunal: A Critically Endangered Medicinal Herb

An efficient micropropagation protocol has been developed for Withania coagulans, a highly endangered medicinal herb and an important natural source of withanolides. Prolific multiplication of axillary buds occurred from the nodal segments taken from adult plant, and cultured on MS medium enriched with BA (0.5 mg l^?1), Kn (0.5 mg l^?1) and PG (0.5 mg l^?1). Nodal segments and shoot tips of elongated microshoots also behaved the same way in cultures and formed multiple shoots through axillary bud multiplication. Addition of PG (0.5 mg l^?1) in the regeneration medium significantly improved induction and elongation of shoot buds. Elongated shoots were placed on filter paper bridges soaked in MS medium with CC (10 mg l^?1) and PG (0.5 mg l^?1) for the initial 7 days’ pulse treatment and thereafter, they were transferred to rooting medium containing IBA (0.25 mg l^?1) + PAA (0.5 mg l^?1) + CC (2 mg l^?1). This protocol has the capacity of producing 1000 plants from one nodal segment after 4 subcultures of 2 weeks each.     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

Transformation of <Emphasis Type="Italic">Liquidambar formosana</Emphasis> L. via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> using a mannose selection system and recovery of salt tolerant lines

Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l⁻¹ induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l⁻¹ α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant (14.6) was observed on MS medium with 3 mg l⁻¹ TDZ, 0.5 mg l⁻¹ NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium with 3 mg l⁻¹ BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with 4 mg l⁻¹ indole-3-butyric acid (IBA) and 1.5 mg l⁻¹ activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro.     

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.     

In Vitro Multiplication of Paedaria foetida L — A Rare Medicinal Plant

Paedaria foetida L, which has great medicinal value is facing danger of extinction. For its conservation, in vitro multiplication may prove one of the best techniques. Micropropagation of P. foetida has been achieved through the culture of nodal explants. The explants produced shoots on MS medium with 0.8% agar. This medium, however, caused high degree of browning of the explants and death of sprouted shoots. Agar free liquid MS medium with polyvinylpyrrolidone (PVP) proved better. Maximum shoot proliferation, free from callus and vitrification but with poor rooting could be obtained in liquid MS medium with PVP (0.8%), NAA (0.5 mg l^?1) and BA (2.0 mg l^?1). The best rooting occurred on semisolid MS medium containing 0.8% agar and 0.5 mg l^?1 IBA.     

In vitro differentiation and plant regeneration of Albizia chinesis (Osb.) Merr

Summary Petiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoog's (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l⁻¹) and indole-3-acetic acid (IAA) (0.5 mg l⁻¹). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l⁻¹) and subsequent subculture in IBA-free medium.     

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 leaf explants of mature sandalwood (Santalum album L.) trees under in vitro conditions

Sandalwood (Santalum album L.) is a small evergreen, hemi-parasitic tree having more than 18 woody species that are mostly distributed in South Asia, Australia, and Hawaii. Its economical importance is derived from its heartwood oil, but its difficult propagation makes conservation essential. The percentage of seed germination is poor and germination time exceeds 12 mo. Vegetative propagation can be accomplished by grafting, air layering, or with root suckers, but the production of clones is inefficient and time consuming. In this study, efficient plant regeneration was achieved via indirect organogenesis from callus cultures derived from leaf tissues of S. album. Callus induction was induced when leaf explants were cultured on woody plant media (WPM) supplemented with either thidiazuron (TDZ) or 2,4-dichlorophenoxyacetic acid. The highest callus frequency (100%) was obtained when leaf tissue was cultured in the medium with 0.4 mg?l^?1 TDZ. Fresh weight (141.92 mg) and dry weight (47 mg) of leaf-derived callus were highest in the medium supplemented with 0.8 mg?l^?1 TDZ. The WPM medium supplemented with 2.5 mg?l^?1 BA?+?0.4 mg?l^?1 NAA was the most effective, producing the highest number of shoot buds (24.6) per callus. The highest number of shoots per explant (20.67) and shoot length (5.17 cm) were observed in media supplemented with 5.0 mg?l^?1 BA and 3.0 mg?1^?1 Kn, respectively. Plantlets were rooted on WPM medium with different concentrations of indole-3-butyric acid (IBA). The highest rooting percentage (91.67) and survival were achieved using WPM media with 1.5 mg?l^?1 IBA. All plantlets survived acclimatization, producing healthy plants in the greenhouse. The current investigation showed efficient in vitro regeneration capabilities of S. album from leaf explants.     

Micropropagation, <Emphasis Type="Italic">in vitro</Emphasis> flowering,and tuberization in <Emphasis Type="Italic">Brachystelma glabrum</Emphasis> Hook.f., an endemic species

Brachystelma glabrum Hook.f. is an endemic plant species of Eastern Ghats, India. In this study, efficient protocols for in vitro micropropagation, flowering, and tuberization of this plant were developed. Sterilized shoot tip and nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs) and additives for shoot induction and multiplication. Both shoot tip and nodal explants showed the best response (90 and 100%, respectively) on MS medium supplemented with thidiazuron (TDZ) at 1.0 mg L^?1. The microshoots multiplied best on MS + TDZ (1.0 mg L^?1) in combination with α-naphthaleneacetic acid (NAA) at 0.5 mg L^?1 and coconut water (CW) at 25%. The highest number of in vitro flowers (4.0 flowers per microshoot) was observed on MS medium supplemented with a combination of N6-benzyladenine (BA) and indole-3-butyric acid (IBA), each at 1.5 mg L^?1. In vitro-derived shoots produced aerial tubers on MS + TDZ (2.0 mg L^?1) + IBA (0.5 mg L^?1) and basal tubers on MS + TDZ at 2.0 mg L^?1. In vitro shoots were best rooted on half-strength (½) MS + NAA at 0.5 mg L^?1. The rooted plantlets were successfully acclimatized in pots with 70% survival after a hardening period of 1 mo. This protocol provides an effective method for the conservation of this endemic plant species.     

Efficient Plant Regeneration via Shoot Tip Explant in Jatropha curcas L

An efficient regeneration method via shoot tip explant has been developed for Jatropha curcas, which is a medicinally as well as economically important plant. Shoot tips were proliferated on MS medium incorporated with BAP (2.0 mg l^?1) and IAA (0.5 mg l^?1) along with adenine sulphate, glutamine and activated charcoal. In vitro produced shoots were induced to root on IBA (0.5–5.0 mg l^?1) added to half strength MS medium. The highest frequency of root induction was on the medium with 3.0 mg l^?1 IBA. Regenerated plantlets were successfully transferred to field after initial acclimatization.     

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.     

TDZ-induced high frequency plant regeneration through multiple shoot formation in witloof chicory (<Emphasis Type="Italic">Cichorium intybus</Emphasis> L.)

A very efficient and rapid regeneration system via multiple shoot formation was developed for Cichorium intybus L. when leaf explants excised from sterile seedlings were cultured on medium supplemented with different concentrations and combinations of various plant growth regulators. In a comparison of leaf lamina and petiole explants, lamina explants produced over three times more shoots than petiole explants, with a mean of 7.5 shoots compared to 2.4. Of the combinations of KIN/IAA, KIN/NAA, BAP/IAA, or BAP/NAA, 0.5 mg l⁻¹ KIN combined with 0.3 mg l⁻¹ IAA was the most effective, producing a mean of 19.7 shoots per lamina explant while the control treatment involving no plant growth regulators produced no shoots at all. When either cytokinin was used alone, BAP was found nearly twice more successful than KIN. However, the most effective treatment of all was the combination of 0.01 mg l⁻¹ TDZ and 1.0 mg l⁻¹ IAA, producing as many as 35.8 shoots per lamina explant. This rate of shoot regeneration is remarkably higher than those previously reported for C. intybus, most likely due to the highly inductive effect of TDZ, which was tested for the first time in this species. Rooting of the shoots was readily achieved on medium containing different concentrations of IAA or IBA. IAA was more effective than IBA and resulted in the highest frequency of shoots that rooted (100%) and mean number of roots per shoot (4.2) when used at 0.5 mg l⁻¹. Hardening off process resulted in a production of more than 80% healthy plantlets.     

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.