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 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.     

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.     

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.     

Callus Induction in Small Flowered Willow Herb (<Emphasis Type="Italic">Epilobium parviflorum</Emphasis> Schreb)

In order to determine the most suitable in vitro tissue culture and plant regeneration conditions for the small flowered willow herb (Epilobium parviflorum Schreb), various explants were cultured on semi-solid MS media containing factorial combinations of plant growth regulators. Callus induction from hypocotyl, cotyledon, petiole and leaf explants was achieved on media containing 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (KIN). All other growth regulator combinations [□-naphtaleneacetic acid (NAA) ± benzylaminopurine (BAP), NAA ± thidiazuron (TDZ), indol acetic acid (IAA) ± Zeatin (ZEA)] tested failed to respond. The best results with cotyledon- and petiole- derived callus were obtained from MS medium supplemented with 1.0 mg l^?1 2,4-D + 0.1 mg l^?1 KIN and 2.0 mg l^?1 2,4-D + 0.2 mg l^?1 KIN. It was observed that B5 basal medium was more effective than MS basal medium for producing seedling and the most effective seed sterilizing solution was 25 % (v/v) sodium hypochlorite (NaOCl). No plant regeneration was observed in either callus induction or during the subculturing stage. This is the first report on in vitro tissue culture study within the genus Epilobium.     

Micropropagation of olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) and application of mycorrhiza to improve plantlet establishment

Micropropagation methods were developed for the three French varieties of olive (Olea europaea L.), Aglandau and Tanche, that have the Appelation d’Origine Contrôlée (AOC) status and one ecotype (05300, Laragne, France). Explants consisting of partially lignified nodal segments were collected from rejuvenated glasshouse-grown plant material. Nodal explants with axillary buds were cultured on different media. For AOC varieties, olive medium modified (OM mod) to contain half the concentration of macroelements was the most efficient in inducing bud break and growth when supplemented with 30 g l^?1sucrose and 4 mg l^?1 zeatin. The resulting shoot buds were further multiplied and maintained on OM mod medium. Rooting was best achieved on OM supplemented with 4 mg l^?1 indole-3-butyric acid (IBA). For the Laragne ecotype, maximal shoot proliferation occurred when explants were cultured on woody plant medium supplemented with 15 g l^?1 sucrose and 0.1 mg l^?1 zeatin. Efficient rooting was achieved with 1 mg l^?1 IBA combined with 0.75 mg l^?1 naphthaleneacetic acid. After acclimatization in the glasshouse, survival rates ranged from 57 to 92%, depending on the genotype. Inoculation of Laragne ecotype microplantlets with the arbuscular mycorrhizal fungus Glomus mosseae significantly improved plant survival and subsequent plant development and growth.     

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.     

Biotransformation of Externally Added Vanillin Related Compounds by Multiple Shoot Cultures of Vanilla planifolia L

The multiple shoots and callus cultures of Vanilla planifolia obtained from the nodal explant on MS medium supplemented with 6-benzylaminopurine (BAP) 2 mg l^?1 and α-naphthalene acetic acid (NAA) 2 mg l^?1 were maintained by regular subculturing every 30 days and also cultured liquid MS medium of the same hormonal combination. Shoots were transferred to the MS basal medium for rooting. Different explants along with vanilla pods and in vitro cultures were analyzed using HPLC for the presence of vanillin and related compounds. When the amount of these compounds was determined in explants and in in vitro cultures after precursor feeding and curing process, explants showed different profile after precursor feeding and after undergoing curing process. During further investigations we have applied a novel approach for curing in vitro tissues as done for vanilla beans. Curing of in vitro shoots resulted in a significant change in the aromatic compound profile.     

Simple and Effective Regeneration of Mungbean (Vigna radiata (L) Wilczek) using Cotyledonary Node Explants

Mungbean (Vigna radiata (L) Wilczek cv ML — 267) is a recalcitrant grain legume species. Direct multiple shoots were developed from the cotyledonary node explants of 2-day-old in vitro grown seedlings of mungbean. Maximum number of shoots (an average 12.1 shoots per explant) was obtained on a medium containing MS salts, B5 vitamins and 5.0 mg l^?1 BAP. A medium with lower BAP concentration appeared suitable for rapid shoot elongation. The elongated shoots were rooted on 0.2 mg l^?1 NAA. The rooted plants were acclimatized under field conditions. The survival of the plants in the greenhouse was 90 %. Plants flowered and set seed normally.     

Induction of callus and plant regeneration in Vicoa indica

Callus cultures were initiated from the stem and leaf explants of aseptically grown Vicoa indica. A simple method is described for plant regeneration from callus and the rapid multiplication of the plants thus obtained. Callus initiation was optimum in Gamborg B5 (B5) basal medium containing either 2.0 mg l^-1 naphthaleneacetic acid (NAA) with 0.2 mg l^-1 kinetin (Kn) or 2.0 mg l^-1 6-benzylaminopurine (BAP) with 0.2 mg l^-1 NAA. The calli initiated on B5 medium were able to proliferate on both Murashige and Skoog (MS) and B5 basal medium. Shoot primordia were obtained from greenish callus on passage to B5 basal medium containing 3.0 mg l^-1 BAP and 1.0 mg l^-1 Kn. On further subculture onto B5 medium containing 0.2 mg l^-1 Kn the shoot primordia developed into plantlets.     

In Vitro Regeneration of a High Oil-yielding Variety of Safflower (Carthamus tinctorius var HUS-305)

A protocol for regular in vitro regeneration of Carthamus tinctorius var HUS-305 is described. The morphogenic response of seed explants and explants from seedlings of different ages were studied on Murashige and Skoog’s medium (MS) supplemented with different growth regulators. The protocol finally standardized involves culture of cotyledonary explants from 2 cm long, 2- to 6-day-old seedlings on MS supplemented with 1.0 mg l^?1 BAP and 0.1 mg l^?1 kinetin. Various other adjuvants viz., adenine sulphate, glutamine and casein hydrolysate were also tested. None of these promoted the caulogenic response significantly. The microshoots could be rooted on medium supplemented with different auxins. The highest rhizogenic response was on 1/2 MS supplemented with 0.2 mg l^?1 NAA.     

Requirements for plant regeneration from protoplasts of the shrubby ornamental honeysuckle,Lonicera nitida cv. Maigrun

Leaf protoplasts of axenic shoot cultures of Lonicera nitida cv Maigrun underwent sustained division to give multicellular colonies (microcalli) on a modified, ammonium-free MS (Murashige & Skoog) medium containing 0.5 mg l^-1 NAA (1-naphthaleneacetic acid), 1.0 mg l^-1 BAP (6-benzylaminopurine) and 150 mg l^-1 casein enzymatic hydrolysate. Callus was produced upon transfer of cell colonies to MS medium with 2.0 mg l^-1 NAA and 0.2 mg l^-1 BAP. About 110 days from isolation protoplast-derived shoots were regenerated on a half-strength MS medium with 0.01 mg l^-1 NAA, 5.0 mg l^-1 BAP, 0.5 mg l^-1 zeatin and a complex mixture of group B vitamins. The replacement of such mixture by 250 mg l^-1 casein enzymatic hydrolysate promoted rhizogenesis in calli, with shoot buds being subsequently regenerated from the protoplast-derived roots. Micropropagation of protoplast-derived shoots (of either origin) was difficult, due to a strong apical dominance, but could be accomplished by transferring single-node explants to half-strength MS medium with 1.5 mg l^-1 BAP. Such shoots were, in turn, successfully rooted and transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - CPW Power et al. (1989) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - F.P.E. final plating efficiency - f.wt. fresh weight - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-butyric acid - I.P.E. initial plating efficiency - MES 2-N-morpholinoethane sulfonic acid - M.P.E. intermediate plating efficiency - MS Murashige & Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PVP-10 polyvinylpirrolidone - Av MW 10,000, TIBA 2,3,5-tri-iodobenzoic acid - Z zeatin     

Clonal Propagation and Production of Genetically Uniform Regenerants from Axillary Meristems of Adult Bamboo

The axillary bud-break and multiple bud induction were obtained from the nodal explants of field-grown culms of Bambusa tulda in liquid Murashige and Skoog’s (MS) basal medium supplemented with 2.0 mg l^?1 6-benzylaminopurine (BAP), 1.0 mg l^?1 kinetin (Kn) and 8% coconut water. Multiple shoots regenerated and proliferated in the liquid MS medium fortified with 3.0 mg l^?1 indolebutyric acid (IBA). While, in B. balcooa, MS medium supplemented with 2.5 mg l^?1 BAP and 1.0 mg l^?1 Kn induced axillary bud-break, bud multiplication and subsequently shoot elongation was obtained after three passages in the same medium. A clump with at least three shoots of both these bamboo species was used as propagule for successful root induction in half-strength MS liquid basal medium supplemented with 0.2 mg l^?1 IBA. Sympodial type of microrhizomes developed in B. tulda and the regenerants acclimatized in the soil easily. Explants collected in the month of October produced best in vitro regeneration response in these two bamboo species. Endogenous phenol content proved detrimental for efficient shoot regeneration. The clonal fidelity of the regenerants was established by RAPD analysis advocating clonal propagation through axillary meristem culture of B. balcooa and B. tulda is reliable for commercial exploitation.     

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.     

Organogenesis and embryogenesis from callus cultures of Azadirachta excelsa

ABSTRACT

Callus production from leaf explants of Azadirachta excelsa (Jack) Jacobs was favoured by Murashige and Skoog medium supplemented with 4 mg l^-1 indole-3-butyric acid (IBA) and 1 mg l^-1 6-benzyladenine (BAP). Increasing the concentration of BAP to 2 mg l^-1 induced shoot regeneration. Adding polyvinylpyrrolidone (PVP) to the medium resulted in a significantly increased number of shoots. Transfer onto medium containing 0.5 mg l^-1 BAP, 0.4 mg l^-1 gibberellic acid (GA₃) and 2% sucrose stimulated elongation of the internodes; subsequent transfer onto medium containing 1 mg l^-1 IBA induced root formation. The histological analysis demonstrated that organogenesis and embryogenesis occurred in the same callus. However, shoots originated inside the callus mass, whereas the embryos originated on the surface. Given that the embryos did not develop beyond the globular or heart-shaped stage, we concluded that the plants regenerated from callus were derived only from organogenesis.     

Endogenous auxin level is a critical determinant for in vitro adventitious shoot regeneration in potato (Solanum tuberosum L.)

Endogenous levels of indole-3-acetic acid (IAA) were determined in the internodal explants of Indian tetraploid potato cultivars (cvs) viz., Kufri Sutlej (K.Sutlej) and Kufri Giriraj (K.Giriraj). Seven fold higher level of endogenous IAA was recorded for cv K.Sutlej over cv K.Giriraj. As a result, perhaps there was a callusing response from the cut end of the internodal explants of both the cvs in the MS basal medium. The extent of callusing was relatively higher in cv K.Sutlej when compared to that in K.Giriraj. The callusing response was inhibitory to shoot morphogenesis. Inclusion of an established anti-auxin, 2,3,5-tri-iodobenzoic acid (TIBA) in the regeneration medium facilitated a high frequency adventitious shoot regeneration response with lower cytokinin levels. Murashige and Skoog (MS) medium containing TIBA at 2.5?mg?l^?1 and 0.25?mg?l^?1 zeatin evoked a 100% regeneration response (4.5 shoot buds per explant) in cv K.Sutlej within 20?C25?days. However, in cv K.Giriraj, which had lower levels of endogenous IAA, 80% regeneration response (1.4 shoot buds per explant) was recorded in an extended period of 40?C45?days on a medium containing 0.5?mg?l^?1 TIBA and 0.1?mg?l^?1 zeatin. Although, TIBA and zeatin induced shoot bud formation, it failed to support sustained growth of the regenerated shoots in cv K.Giriraj. Hence, 0.01?mg?l^?1 ??-naphthaleneacetic acid (NAA) with a relatively higher concentration of zeatin (1.0?mg?l^?1) were used for sustained shoot regeneration (3.3 shoot buds per explant) within 25?days. From our results, it is evident that there was a difference in the requirement of exogenous zeatin levels required to induce a regeneration response in two cultivars of potato and this is attributed to the variable levels of endogenous IAA.     

High frequency plantlets regeneration from seedling explants of Prosopis tamarugo

Callus cultures of Prosopis tamarugo Phil (Leguminosae, Sub family-Mimosoideae) were established from hypocotyls and cotyledons on MS medium supplemented with NAA (2.0 mg l^-1) and BAP (0.2 mg l^-1). Regeneration through various juvenile explants was obtained on hormone-free and high cytokinin containing Murashige and Skoog's medium. Multiple shoot buds formation was observed from the embryonic axis on MS medium incorporated with BAP (5.0 mg l^-1)). Elongation of shoot buds was observed on subsequent transfer to MS medium with BAP (1.0–2.5 mg l^-1) or without BAP. Explants containing apical meristem showed higher number of shoot formation at an early period. De novo shoot buds formation through callus morphogenesis was observed at the base of differentiated shoots on high cytokinin containing medium. All the manipulations of salt strength of MS, nitrogen, carbon, ascorbic acid and polyamines failed to induce organogenesis in isolated callus. In vitro produced shoots were rooted on MS medium supplemented with IBA or NAA singly or in combination.Abbreviations HC high cytokinin (BAP 5.0 mg l^-1) - BAP 6-benzyl amino purine - IBA indole-3-butyric acid - HF hormone free - NAA I-naphthalene acetic acid - MS Murashige & Skoog     

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.     

Micropropagation of Capparis decidua through In Vitro Shoot Proliferation on Nodal Explants of Mature Tree and Seedling Explants

In vitro clonal propagation of Capparis decidua was achieved using nodal explants from mature trees, and cotyledonary node, cotyledon and hypocotyl explants taken from the seedlings. Explants cultured on MS medium supplemented with BAP showed differentiation of multiple shoots and shoot buds in 4–5 weeks in the primary cultures. The medium with BAP (5 mg/l) was the best for shoot bud proliferation from the nodal as well as seedling explant. Shoot multiplication was best on cotyledonary node. In the nodal explants shoot multiplication was best on medium supplemented with 5 mg/l BAP and after second subculturing further multiplication of shoot buds was highest on the medium containing 3 mg/l BAP. Shoots were separated from mother cultures in each subculturing for rooting. Rooting was best achieved using 1 mg/l IBA in the medium. Rooted plantlets were transferred td earthen pots with garden soil and peat moss mixture.     

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.