Morpho-histological characterization and direct shoot organogenesis in two types of explants from <Emphasis Type="Italic">Bacopa monnieri</Emphasis> on unsupplemented basal medium

In the present study, high frequency regeneration has been obtained via de novo direct shoot organogenesis from leaf and internode explants in Murashige and Skoog (MS) basal medium without any phytohormone supplementation in Bacopa monnieri, an indigenous traditionally used medicinal herb. Leaves and internodes from different positions were excised from 4-weeks-old in vitro propagated B. monnieri plants and cultured on MS basal medium supplemented with 3% (w/v) sucrose and 0.75% (w/v) agar for 4 weeks. The induction of de novo shoot buds was observed at petiolar cut edges of leaf and both proximal and distal cut ends of internode explants within 10–15 days of culture. The first histological changes could be observed after 4–5 days, with meristematic activity of vascular bundles. Proliferation of epidermal cells gave rise to dome-shaped protuberances followed by shoot apical meristems formation and their vascular connections with explant tissues within 2 weeks of culture. However, a basipetal gradient of shoot regeneration from both types of explants collected along the branch axis was noticed after 4 weeks of culture. Leaf and internode explants near the basal region exhibited significantly higher number of shoot buds and micro shoots (8.8/leaf explant and 15/internode explant). Microshoots (7–12 micro shoots/leaf or internode explants) elongated (shoot length 8–9 cm) within 8 weeks on phytohormone free MS medium. Excised micro shoots rooted (100%) in hormone free MS medium within two weeks of culture. Rooted plants were then acclimatized and transferred to field with 95% survival. This protocol may be used for micropropagation, genetic transformation as well as a model system for evaluation of changes associated with acquisition of competence of differentiated cells in phytohormone free medium.     

Micropropagation of <Emphasis Type="Italic">Pongamia pinnata</Emphasis> through enhanced axillary branching

A complete protocol is presented for the first time for the micropropagation of Pongamia pinnata, a biofuel tree, using cotyledonary nodes derived from axenic seedlings. Multiple shoots were induced in vitro from nodal segments through forced axillary branching. Murashige and Skoog (MS) medium supplemented with 7.5 μM benzylaminopurine (BAP) induced up to 6.8 shoots per node with an average shoot length of 0.67 cm in 12 d. Incorporation of 2.5 μM gibberellic acid (GA₃) in the medium during the first subculture after establishment and initiation of shoot buds significantly improved the shoot elongation. Single use of GA₃ during the first subculture eliminated the need for prolonged culturing on BAP medium. Further use of GA₃ in the medium was not useful. Shoot culture was established for at least two subcultures without loss of vigor by repeatedly subculturing the original cotyledonary node on shoot multiplication medium followed by shoot elongation medium after each harvest of the newly formed shoots. Thus, from a single cotyledonary node, about 16–18 shoots were obtained in 60 d. Shoots formed in vitro were rooted on full-strength MS medium supplemented with 1.0 μM indole butyric acid (IBA). Plantlets were successfully acclimated, established in soil, and transferred to the nursery.     

Efficient shoot and root formation from cotton shoot apices

Successful shoot and root induction were obtained from shoot apices of two cotton (Gossypium hirsutum L.) genotypes, Nazilli 84S and Çukurova 1518, which are widely planted in Turkey. Plant tissue culture systems were established on Murashige and Skoog (MS) medium supplemented with various plant growth regulators using seven-day-old shoot apices as explants. The shoot apex size was of 2–3 mm; it contained the meristem and unexpanded leaves. Shoot apices were placed on MS plus vitamins and combinations of various plant hormones. The best regeneration responses were obtained for cv. Nazilli 84S (98%) on MS + 0.1 mg/l kinetin (KIN) + 1 g/l polyvinylpyrrolidone (PVP) and for Çukurova 1518 (94%) on MS + 0.1 mg/l KIN + 2 mg/l NAA + 1 g/l PVP. Including germination, all regeneration and rooting processes lasted only 5 weeks. The shoot apices of both genotypes developed successfully without intervening callus formation, and no significant differences between cultivars were found. All regenerated plants of both genotypes were phenotypically normal and set seeds. This shoot meristem-based rapid regeneration method can also be used in the cases of biolistic and Agrobacterium-mediated transformation.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> by organogenesis and somatic embryogenesis

In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 100 mg dm⁻³ glutamine, 20.9 μM N ⁶-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing 2 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous proline content up to 28^th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium with 3 % (m/v) sucrose, 100 mg dm⁻³ myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine.     

TDZ induces shoot regeneration in various Kalanchoë blossfeldiana Poelln. cultivars in the absence of auxin

In order to optimize shoot regeneration in Kalancho? blossfeldiana, leaf and internode explants of seven cultivars including one inter-specific were studied. The effects of various combinations of α-naphthalene acetic acid (NAA) (0, 0.57 M) and thidiazuron (TDZ) (0, 0.45, 4.5, 22.5, 67.5 μM) on MS medium were examined. In all cultivars shoot regeneration frequency and number of shoots per explant were enhanced by increasing TDZ concentration. Supplementing the media with NAA did not improve shoot regeneration. Maximum regeneration frequency and optimum concentration of TDZ for shoot regeneration depended significantly on the cultivar. Internode explants, but not leaf explants, of some cultivars, were able to produce adventitious shoots without treatment with growth regulator.     

Allelopathic effect of aqueous extracts of three weed species on the growth and leaf chlorophyll content of bread wheat

The purpose of this study was to evaluate the allelopathic effect of weeds (Avena fatua, Melilotus officinalis and Polypogon hissaricus) on germination, growth, dry biomass and chlorophyll concentration of three cultivars of wheat (Ata Habib, Pirsabaq and Serin). In germination test, different concentrations of aqueous extracts (5, 10 and 15?g/l) of the three weeds significantly reduced percent germination; however, 15?g/l extract of M. officinalis resulted in complete failure of germination of cultivar Pirsabaq. In pot culture, root and shoot length, chlorophyll concentration and seedling dry biomass of the three wheat varieties showed differential responses to different weeds. Aqueous extract at 15?g/l of A. fatua increased root and shoot length and dry biomass of cultivar Pirsabaq; however, these parameters were significantly retarded in other two wheat cultivars by extract of weeds. Moisture content of the cultivars did not show any response to allelopathic stress of the weeds. In contrast, chlorophyll concentration in Pirsabaq and Serin was significantly increased by aqueous extract of all the weeds but reduced it in cultivar Ata Habib by 50%. In general, Ata Habib was found to be the most sensitive cultivar to the imposed allelopathic stress. The phytotoxic potential of three weeds was found in the order of A. fatua?>?M. officinalis?>?P. hissaricus.     

Plantlet Regeneration via Multiple Shoot Induction in Indian Cultivars of Lucerne (Medicago sativa L)

An efficient protocol has been developed for in vitro plant regeneration via multiple shoot induction in lucerne (Medicago sativa L). Shoot tips from in vitro grown 5–6 days old seedlings of 3 cultivars, LLC-3, Chetak and RL-88 were used as explants for multiple shoot induction on MS medium supplemented with cytokinins. Maximum of 14 shoots per apical meristem were observed in case of cv Chetak on MS medium supplemented with BAP (12.6 μM) and KN (9.3 μM). Shoot elongation on MS medium supplemented with GA (5.8 μM), while root induction was achieved on MS medium supplemented with IAA (11.4 μM) and activated charcoal (2.0 g l^?1). Tissue raised plants showed 75% survival after transfer to soil under field conditions.     

Nutritional, growth, and reproductive responses of maize (Zea mays L.) to arbuscular mycorrhizal inoculation during and after drought stress at tasselling

The effects of root colonization by the arbuscular mycorrhizal (AM) fungus Glomus intraradices Schenck & Smith on nutritional, growth, and reproductive attributes of two tropical maize cultivars with different sensitivities to drought were studied. Freshly regenerated seeds of selection cycles 0 (cv. C0, drought-sensitive) and 8 (cv. C8, drought-resistant) of the lowland tropical maize population "Tuxpeño sequía" were used in this greenhouse experiment. Maize plants were subjected to drought stress for 3 weeks following tasselling (75–95 days after sowing) and rewatered for the subsequent 5 weeks until harvest. Mycorrhizal (M+) plants had significantly higher uptake of N, P, K, Mg, Mn, and Zn into grain than non-mycorrhizal (M–) plants under drought conditions. AM inoculation also produced significantly greater shoot masses in C0 and C8 regardless of the drought-stress treatment. In the sensitive cultivar C0, drought stress reduced the shoot mass and grain yield by 23% and 55%, respectively, when roots were not colonized, while the reductions were only 12% and 31%, respectively, with mycorrhizal association. In addition, the emergence of tassels and silks was earlier in M+ plants than in M– plants under drought conditions. Mycorrhizal response was more pronounced under both well-watered and drought conditions in C0 than in the C8 cultivar. The overall results suggest that AM inoculation affects host plant nutritional status and growth and thereby alters the reproductive behaviour of maize under drought conditions.     

An improved protocol for micropropagation of Mallotus repandus (Willd.) Müll.Arg.

Node and internode explants of Mallotus repandus were precultured on basal medium (BM: Murashige and Skoog (MS) medium with 3% sucrose and 0.55% Agargel) for 0–18 d before culture on shoot induction Medium (SIM: BM added with 4.44 μM of benzylaminopurine) for 4 wk. The cultures were subsequently transferred to BM for 4 wk for shoot elongation. Node explants precultured on BM for 14 d before incubation on SIM were at an optimum for shoot regeneration with the response rate of 95%, compared to a 21% response for the control without preculture. Internode explants precultured on BM for 16 d responded with an optimal shoot formation response rate of 69%, whereas the control response rate was 6%. The maximum shoot regeneration rates were 3.1 ± 0.3 and 2.7 ± 0.4 shoots/responding explant in node and internode explants, respectively. This study demonstrates for the first time that shoot organogenesis can be induced from internode explants of M. repandus. Furthermore, the results suggest that the explants need to acquire competence before shoot organogenesis. Rooting was obtained by incubation of regenerated shoots on half-strength MS with 10.74 μM of 1-naphthylacetic acid for a week before culture on half-strength MS for 4 wk. Regenerated plants were successfully transferred to soil.     

In vitro organogenesis from internode derived callus cultures of Capsicum annuum L.

An efficient protocol of shoot organogenesis and plant regeneration from internode derived callus has been developed for Capsicum annuum. Optimal callus was developed from internodal segments on Murashige and Skoog (MS) medium supplemented with 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 2.0 μM 6-benzyladenine (BA). Shoot differentiation was achieved from the surface of callus when transferred on shoot induction medium containing BA and thidiazuron (TDZ) alone or in combination. The highest number of de novo adventitious shoots (25.4?±?1.42) and shoot length (4.6?±?0.37 cm) was recorded on MS medium supplemented with 5.0 μM BA and 2.5 μM TDZ. The individual elongated shoots were rooted well on MS medium supplemented with 1.0 μM Indole-3-butyric acid (IBA). The in vitro raised plantlets with properly developed shoot and roots were acclimatized successfully and grew well in the greenhouse. All the regenerated plants appeared normal with respect to morphology and growth characteristics with 85% survival rate.     

Factors affecting adventitious regeneration from in vitro leaf explants of 'Improved French' plum, the most important dried plum cultivar in the USA

An adventitious shoot regeneration protocol from in vitro leaves of the most important dried plum cultivar in the USA, ‘Improved French’, has been established. Factors affecting regeneration were studied in order to optimise regeneration. The proliferation medium in which the shoots, used as the source of leaf explants, were cultured had a strong influence on subsequent regeneration. Shoot regeneration was observed at a mean frequency of 52% when a Murashige-based and Skoog-based shoot culture medium with 3 μM N⁶-benzylaminopurine and 0.25 μM indole-3-butyric acid (IBA) was employed compared with shoot regeneration frequencies of less than 5% for a Quoirin-based and Lepoivre-based shoot culture medium, with 8.9 μM N⁶-benzylaminopurine and 0.49 μM IBA. The shoot regeneration medium contained α-naphthaleneacetic acid at 2.0–6.0 μM and thidiazuron at 4.5–15.0 μM. 2,4 Dichlorophenoxy-acetic acid at 9.0 μM was included in the medium but only for the first 4 days of culture. Shoot regeneration frequencies were positively related to thidiazuron concentration and significantly greater (P < 0.05) for 9–15 μM thidiazuron than for the media with 4.5 μM thidiazuron. Leaf explants, incubated in a 16-h-light/8-h-dark photoperiod or in the dark for 1 week followed by exposure to light, showed significantly more organogenic activity (P < 0.01) than was observed for leaves cultured in the dark for 2 or 3 weeks before they were transferred to the light. The utilisation of Bacto agar (0.7%) as the gelling agent increased organogenesis compared with media gelled with TC Agar (0.7%), or an agar–gellan gum blend (Agargel™) (0.45%). The addition of the ethylene inhibitor silver thiosulphate at 60–120 μM also improved organogenesis. When all the studied factors were optimised, a regeneration rate of 65% was achieved. Rooting frequency of regenerated shoots was significantly increased (P < 0.05) by the use of full-strength Murashige and Skoog salts (40%) or 100 mg L⁻¹ phloroglucinol (53%) to the rooting medium.     

An efficient,widely applicable cryopreservation of Lilium shoot tips by droplet vitrification

We report a straightforward and widely applicable cryopreservation method for Lilium shoot tips. This method uses adventitious shoots that were induced from leaf segments cultured for 4 weeks on a shoot regeneration medium containing 1 mg/l α-naphthaleneacetic acid and 0.5 mg/l thidiazuron. Shoot tips (1.5–2 mm in length) including 2–3 leaf primordia were precultured on Murashige and Skoog (MS; 1962) medium with 0.5 M sucrose for 1 day and then treated with a loading solution containing 0.4 M sucrose and 2 M glycerol for 20 min, followed by a Plant Vitrification Solution 2 (PVS2) treatment for 4 h at 0 °C. Dehydrated shoot tips were transferred onto 2.5 µl PVS2 droplets on aluminum foil strips, prior to a direct immersion into liquid nitrogen for 1 h. Frozen shoot tips were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for shoot regrowth. Shoot regrowth levels ranged from 42.5 % for L. longiflorum × Oriental ‘Triumphator’ to 87.5 % for L. Oriental hybrid ‘Siberia’, with a mean shoot regrowth level of 67.1 % across the six diverse Lilium genotypes tested. Histological observations found that the survival patterns were similar in cryopreserved shoot tips of ‘Triumphator’ and ‘Siberia’. Assessments using inter-simple sequence repeat markers found no differences in regenerants recovered from the control stock cultures and from cryopreserved shoot tips in ‘Triumphator’ and ‘Siberia’. This Lilium droplet-vitrification cryopreservation method is efficient, simple and widely applicable for the long-term conservation of lily genetic resources.     

Influence of Four Nematodes on Root and Shoot Growth Parameters in Grape

Two grape cultivars, susceptible French Colombard and tolerant Rubired, and four nematodes, Meloidogyne incognita, Pratylenchus vulnus, Tylenchulus semipenetrans, and Xiphinema index, were used to quantify the equilibrium between root (R) and shoot (S) growth. Root and shoot growth of French Colombard was retarded by M. incognita, P. vulnus, and X. index but not by T. semipenetrans. Although the root growth of Rubired was limited by all the nematodes, the shoot growth was limited only by X. index. The R:S ratios of Rubired were higher than those of French Colombard. The reduced R:S ratios of Rubired were primarily an expression of reduction in root systems without an equal reduction in shoot growth, whereas in French Colombard the reduced R:S ratios were due to a reduction in both shoot growth and root growth and to a greater reduction in root growth than shoot growth. All nematodes reproduced equally well on both cultivars. Both foliage and root growth of French Colombard were significantly reduced by M. incognita and P. vulnus. Nematodes reduced the shoot length by reducing the internode length. Accumulative R:S ratios in inoculated plants were significantly smaller than those in controls in all nematode treatments but not at individual harvest dates. Bud break was delayed by X. index and was initiated earlier by P. vulnus and M. incognita. All buds in nematode treatments were less vigorous than in controls.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

Copper-stress induced alterations in protein profile and antioxidant enzymes activities in the in vitro grown Withania somnifera L.

Withania somnifera L. seedlings were grown in half-strength MS (Murashige and Skoog) basal medium for 4 weeks and then transferred to full-strength MS liquid medium for 3 weeks. The sustainable plants were subcultured in the same medium but with different concentrations (0, 25, 50, 100 and 200 μM) of Cu for 7 and 14 days. The growth parameters (root length, shoot length, leaf length and total number of leaves per plant) showed a declining trend in the treated plants in a concentration dependant manner. Roots and leaves were analyzed for protein profiling and antioxidant enzymes [catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and guaiacol peroxidase (GPX, EC 1.11.1.7)]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude protein extracts showed the appearance of some new proteins due to Cu treatment. In plant samples grown with 25 and 50 μM of Cu, a rapid increase in antioxidant activities were noticed but at higher concentration (100 and 200 μM) the activities declined. Isoforms of CAT, SOD and GPX were separated using non-denaturing polyacrylamide gel electrophoresis and concentration specific new isoforms were noticed during the study. Isoforms of the antioxidant enzymes synthesized due to Cu stress may be used as biomarkers for other species grown under metal stress.     

Droplet-vitrification cryopreservation of <Emphasis Type="Italic">in vitro</Emphasis>-grown shoot tips of grapevine (<Emphasis Type="Italic">Vitis</Emphasis> spp.)

An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5–6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp.     

Direct shoot regeneration and genetic fidelity analysis in finger millet using ISSR markers

Finger millet (Eleusine coracana (L.) Gaertn.), an economically important food crop is cultivated widely in the arid and semi-arid tropics of Africa and Asia. In the present study, an efficient micropropagation protocol has been established for finger millet genotypes CO 9, CO (Ra) 14 and GPU 28 using shoot apical meristems (SAMs). Shoot proliferation medium (SPM) containing Murashige and Skoog’s (MS) medium amended with 3.0 mg/l 6-benzylaminopurine produced the highest shoot regeneration frequency (86.60%) with an average of 26.45?±?0.34 shoots per explant and 6.26?±?0.38 cm shoot length in CO 9. An increase in the number of shoots per explant was observed when SAMs were repeatedly sub-cultured in SPM at 2 weeks interval for 8 weeks. Rooting of the regenerated shoots was achieved in full-strength MS medium containing indole-3-acetic acid (IAA) or indole-3-butyric acid. Rooting medium containing 0.25 mg/l IAA exhibited highest rooting frequency (100%) with an average root length of 4.44?±?0.15 cm. In vitro rooted shoots transferred to the field conditions resulted in 100% survivability.Genetic fidelity of 3-month old mother plant and micropropagated plantlets was confirmed using 3′-anchored dinucleotide inter simple sequence repeats. A total of 115 amplicons generated for CO 9, CO (Ra) 14 and GPU 28 were monomorphic, revealing no variation among mother plant and micropropagated plantlets. Thus, SAMs could serve as a suitable explant for the mass multiplication of true-to-type plants and genetic transformation in finger millet.     

An improved method for micropropagation and regeneration of date palm (Phoenix dactylifera L.)

We developed a novel large-scale micropropagation pathway for date palm (Phoenix dactylifera L.) based on organogenesis. We obtained organogenic stems from shoot tip explants of the Moroccan date palm cultivar Najda, and investigated shoot proliferation from these organogenic stems in vitro on various media; Beauchesne medium (BM) and Murashige and Skoog medium (MS) at full-strength, half-strength, and one-third-strength, containing various concentrations (0, 0.25, 0.5, and 1 mg/L) of 2-naphthoxyacetic acid (NOAA) and kinetin. The optimal medium during the multiplication phase was half-strength Murashige and Skoog medium (MS/2) supplemented with 0.5 mg/L NOAA and 0.5 mg/L kinetin (23.5 morphologically superior shoots per explant, with low vitrification rates). For the shoot elongation phase, shoots were transferred to the same proliferation medium, or to MS or MS/2 media without plant growth regulators (PGRs). Shoots elongated rapidly and showed a high rate of root formation on media supplemented with PGRs. For example, on MS/2 medium containing 1 mg/L NOAA and 1 mg/L kinetin, the average shoot length was 15.1 cm, the average number of roots per shoot was 6.2, and their average length was 3.4 cm. On PGR-free media, shoots were shorter with wider and greener leaves, and had fewer roots. The plantlets were transferred to a greenhouse for acclimation. The survival rate after 2 months was related to the medium used during the elongation phase; >90 % of shoots that were cultured on PGR-free media survived, while there was a poor survival rate of shoots that had been cultured on media containing PGRs.     

Efficient,one step and cultivar independent shoot organogenesis of potato

An efficient, one step and genotype independent protocol of shoot organogenesis was developed from leaf and internodal explants taken from microshoots of different cultivars of potato (Solanum tuberosum L.). Initially, microshoots were cultured on basal Murashige and Skoog medium additionally supplemented with 10 µM AgNO₃ (MS1 medium) to achieve healthy shoot growth required to get the quality explants. Shoot organogenesis was induced from both types of explants (leaf and internodal) on MS1 medium variously supplemented with 6-benzyladenine (BA) and gibberellic acid (GA₃). Maximum explants were induced shoot organogenesis on MS1 medium supplemented with 10 µM BA and 15.0 µM GA₃ from both the cultivars namely ‘Kufri Chipsona 1’ and ‘Kufri Pukhraj’. Among the types of explants used, better response was observed from internodal segments as compared to leafs. This optimized medium combination was found to be equally effective for all the eight cultivars tested namely ‘Kufri Pukhraj’, ‘Kufri Chipsona 1’, ‘Kufri Chipsona 2’, ‘Kufri Jyoti’, ‘Kufri Surya’, ‘Kufri Chandramukhi’, ‘Kufri Khyati’ and ‘Desiree’. The clonal uniformity of the regenerated shoots was confirmed using random amplified polymorphic DNA and inter-simple sequence repeats markers.     

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.