Micropropagation in Peanut (Arachis hypogaea L.)

Multiple shoots in Arachis hypogaea L. could be induced from the de-embryonated cotyledons (DC), embryo-axes (EA) and mature whole seeds (MWS) in MS medium supplemented with different levels of benzylaminopurine (BAP). DC was the most suitable explant with 57.9 % induction and more than 40 shoots per explant in 31.6 % of cases. Though EA and MWS had high percent induction at or above 30 mg dm^–3 BAP, only 10 – 14 shoots per explant were observed. In DC, multiple shoots were confined to the proximal end and in EA they originated from the axillary bud region. Histological studies on DC confirmed the origin of shoots from the region of attachment with the embryo. Shoots could be rooted in MS medium containing 2 g dm^–3 charcoal and 200 mg dm^–3 casein hydrolysate. Sixty percent of the rooted plantlets could be established in the field.     

Recovering vitrified carnation (Dianthus caryophyllus L.) shoots using Bacto-Peptone and its subfractions

Vitrified shoots regenerated from carnation petals (Dianthus caryophyllus L. cv. Scania) were recovered by culturing them in a medium containing 3.0 g/l Bacto-Peptone. Wax structures not found on vitrified shoots developed on the abaxial surface of leaves of recovered shoots and on those of normal leaves. Recovered shoots were rooted and successfully acclimatized while vitrified shoots could not survive the acclimatization process. The Bacto-Peptone solution was fractionated and the efficiency of each fraction for the recovery of vitrification was examined. Only basic, non high molecular fractions whose molecular weight was less than 10,000 were effective.     

In vitro shoot proliferation and propagation of guava (Psidium guajava L.) from germinated seedlings

Guava seeds were germinated on Murashige and Skoog (MS) medium with or without 8.8 μM benzyladenine (BA). BA increased the rate of germination and the number of lateral shoots (3.4 vs 1.2 per seedling). Stem nodes from these lateral shoots were cultured on proliferation media with 4.4 μM BA, and multiple shoots (3.5) were formed within 4 weeks of culture. Increasing the concentration of BA or the addition of naphthaleneacetic acid (NAA) did not affect shoot formation. Shoots produced from explants and lateral shoots from germinated seedlings were rooted in media containing activated charcoal (AC) or 9.8 μM indolebutyric acid (IBA). Shoots rooted with IBA had a higher rooting percentage (100% vs 75%) and a greater number of roots (5.5 vs 3.2) but the shoots were shorter (2.6 vs 3.4 cm) than when rooted in AC, and they required an additional 4 weeks of culture in media with AC to achieve shoot elongation. About 80% of the shoots with roots survived in the glasshouse and produced normal phenotypic plants.     

In vitro clonal propagation of annatto (Bixa oreliana L.)

Summary A protocol for in vitro propagation of Bixa orellana is described. Plants were regenerated from shoot apex and nodal explants on B5 medium supplemented with 4.9 μM 2-isopentenyl adenine. The multiplication factor of shoot apex explants was higher (nine shoots per explant) than that of the nodal explants (five shoots per explant). Regardless of the position of the nodes, all the nodal explants gave similar responses. However, the size of the nodal explant was an important factor in producing multiple shoots: 0.5 cm nodal explants produced the maximum multiple shoots. Regenerated shoots from shoot apex explants rooted best on MS medium supplemented with 0.05 μM α-naphthalene acetic acid (NAA). whereas shoots regenerated from nodal explants needed 2.7 μM NAA for rooting. Eighty per cent survival of in vivo transferred plants occurred on the best potting substrate, coco peat. Since the multiplication factor was nine per explant, this protocol can be use for commercial microprogation. However, the regeneration capacity declined after 10 subcultures. Approximately, 3350 rooted plants could be generated in 10 mo. after eight subcultures, from one shoot with a shoot apex and four nodes.     

In vitro regeneration of Norway maple (Acer platanoides L.)

Regenerants were produced from axillary buds, but not from petiole segments, greenwood cuttings and leaf discs. Petiole segments and greenwood cuttings responded by massive callus cell proliferation without adventitious shoot formation. The development of induced buds into shoots occurred on WPM medium containing kinetin. Vigorous shoots larger than 2.0 cm in length were successfully rooted in half strength WPM medium supplemented with 1.0 mg dm^-3 indole-3-butyric acid.     

Promotion by phloroglucinol of adventitious root formation in micropropagated shoots of adult wild cherry (Prunus avium L.)

Micropropagated shoots of wild cherry (Prunus avium L.) produced roots in auxin-free medium. Phloroglucinol (PG) increased the proportion of shoots that rooted, while phloretic acid reduced this response in medium with or without PG, and cancelled the promotive effect of PG. Concentration of PG also significantly affected rooting in media with and without auxin. The proportion of shoots rooting in media containing auxin, or auxin plus PG, increased with the number of successive subculture, but the proportion that rooted with PG alone was unaffected by the number of subcultures. Before the shoots had become responsive to auxin, 1 mM PG was more effective than auxin in inducing root formation.     

Shoot regeneration of young leaf explants from basil (Ocimum basilicum L.)

Summary An efficient plant regeneration protocol was successfully developed for basil (Ocimum basilicum L.). Explants from 1 mo. old seedlings yielded the highest frequency of 85% regeneration with an average of 5.1 shoots per explant. The regeneration protocol was performed on three basil varieties (Sweet Dani; methylcinnamate; Green Purple Ruffles). Callus and shoot induction was initiated on Murashige and Skoog basal medium supplemented with thidiazuron (16.8 μM) for approximately 30 d. Shoot induction and development were achieved by refreshing the induction medium after 14 d. The most morphogenetically responsive explants were from the first fully expanded true leaves of greenhouse-grown basil seedlings. All developing bud tissue demonstrated temporary anthocyanin expression; however, anthocyanin expression in Green Purple Ruffles remained stable until maturity. Developing shoots were rooted in the dark on media with thidiazuron removed. Within 20 d, rooted plantlets were transferred and acclimatized under greenhouse conditions where they developed normal morphological characteristics. This is the first report of a successful in vitro regeneration system for basil through primary callus.     

Rapid in-vitro plant regeneration of cotton (Gossypium hirsutum L.)

A rapid, clonal propagation procedure has been developed to regenerate mature cotton (Gossypium hirsutum L.) plants from pre-existing meristems that were excised from in-vitro-grown tissues. This plant regeneration procedure was applicable to diverse cotton germplasms and required specific concentrations of 6-benzylaminopurine (BA) depending on the origin of the meristems. All shoots regenerated directly without a callus phase. Screening BA concentrations (0.0–10.0 μm) demonstrated that shoot meristems (apices), secondary leaf nodes, primary leaf nodes, and cotyledonary nodes derived from in-vitro-grown 28-day-old seedlings (Paymaster HS26) varied in their ability to form elongated shoots depending on the level of BA. Indicative of a germplasm-independent procedure, a BA concentration screen (0.0, 0.3, 1.0 μm) demonstrated that explants with pre-existing meristems, excised from diverse germlines, were also able to form elongated shoots at 0.3 μm BA. In most cases, elongated shoots derived from this procedure were rooted by a two-step process: an in-vitro maturation step (Murashige and Skoog medium-activated charcoal) followed by planting into soil after basal application of Rootone. This BA plant regeneration procedure was rapid, reproducible, and highly efficient for Stoneville 7A, Paymaster HS26, and other high-fiber-yielding germlines. Regenerated plants were phenotypically normal and all of the mature plants regenerated to date have initiated flowers and set viable R₁ seeds. Received: 15 March 1997 / Revision received: 28 August 1997 / Accepted: 5 September 1997     

In Vitro Shoot Regeneration from Callus, Leaf Axils and Petioles of Sugar Beet (Beta vulgaris L.)

Procedures are described for producing axillary shoots fromseedling apices and adventitious shoots from petioles and leaf-derivedcallus of sugar beet cultivars. The rate of adventitious shootregeneration from petioles was influenced by temperature, BAPconcentration of the medium, and the time in culture of theseedling apices from which the petioles were excised. Petiolesectioning confirmed that adventitious shoots originated inthe sub-epidermal parenchyma. Two distinct types of callus wereproduced from leaf explants, but only white friable callus wascapable of shoot development. This callus developed from browntissue and was composed of thin-walled cells with dense cytoplasmand prominent nuclei. Green compact callus with thick-walledlignified cells developed from green tissue, but did not produceshoots. Successful seed sterilization and shoot regenerationfrom petiole explants and callus was cultivar-dependent. Adventitiousshoots were rooted and successfully transplanted to pottingcompost under glasshouse conditions. Key words: Adventitious shoots, axillary shoots, callus, sugar beet (Beta vulgaris L.)     

Improvements in rooting regenerated safflower (Carthamus tinctorius L.) shoots

A continuing obstacle for regenerating safflower (Carthamus tinctorius L.) plants from cultured explants or callus has been a reliable method for rooting shoots. For shoots directly regenerated from primary explants, 76% of shoots rooted after a 7-d exposure to 10 mg/1 indole-3-butyric acid. Auxin source, concentration or exposure time did not greatly affect root formation or morphology, but strongly affected callus production. Shoots infected with Agrobacterium rhizogenes produced massive numbers of fibrous roots, but shoots did not elongate or survive transfer to soil. Shoot hyperhydricity symptoms were reduced by including 1 g/1 activated charcoal in rooting media. The optimal protocol for inducing root formation consisted of a 7-d exposure to 10 mg/l indole-3-butyric acid in root induction media, followed by incubation in media containing 15 g/l sucrose and 1 g/1 activated charcoal for 21 d.Abbreviations IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA anaphthalene acetic acid - POP 2,3,5-trichloro--phenoxypropionic acid     

In Vitro Plantlet Formation in Mangosteen (Garcinia mangostana L.)

Optimum conditions were determined for in vivo growth and multiplicationof Garcinia mangostana L. using explants from aseptically germinatedseedlings and field-grown plants. Proliferating shoots wereobtained from cotyledon segments cultured on modified Murashigeand Skoog's (1962) medium with 6-benzylaminopurine. Juvenileleaf segments produced adventitious buds on Woody Plant Medium(Lloyd and McCown, 1981). Root segments gave few buds. Shoottip, nodal, and internodal explants gave multiple axillary andadventitious buds. Shoots were multiplied by enhanced axillaryand adventitious bud formation. The shoots were rooted withindolebutyric acid treatment. Rooted shoots were readily establishedin vermiculite: sand (1:1) mixture. Garcinia mangostana L., Mangosteen, tissue culture, shoot regeneration, bud development     

Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.)

A protocol is presented for direct adventitous shoot organogenesis and complete plant regeneration from seedling-derived explants of pomegranate (Punica granatum L.), a tropical fruit tree. Murashige and Skoog (1962) (MS) medium enriched with 8.9 mumol/L benzyladenine (BA), 5.4 mumol/L naphthaleneacetic acid (NAA) and 10% coconut water (CW) induced adventitious shoot bud differentiation in axenic seedling-derived cotyledons as well as hypocotyl segments. The cotyledons were more responsive than the hypocotyls. Addition of ethylene inhibitors such as AgNO3 (10-40 mumol/L) and aminoethoxyvinylglycine (AVG) (5-15 mumol/L) to the medium markedly enhanced regeneration frequency as well as number of shoots obtained per explant. The promotive effect of AVG and AgNO3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO3 (20 mumol/L) or AVG (10 mumol/L) induced adventitious shoot buds from 57% or 53% of the cotyledon explants respectively. These shoot buds developed into shoots upon transfer to a regeneration medium without AgNO3 and AVG. The promotive effect of AVG on shoot regeneration was reversed by exogenous application of 20 mumol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. On the other hand, shoot regeneration stimulated by AgNO3 was relatively less affected by CEPA. Regenerated shoots were rooted in half-strength MS medium (1/2 MS) containing 0.54 mumol/L NAA. The well rooted plantlets were acclimatized and eventually established in soil.     

Stimulation of node and lateral shoot formation in micropropagation of olive (Olea europaea L.) by using dikegulac

Dikegulac, a growth regulator, was shown to stimulate in vitro shoot multiplication of olive cultivars Canino, Frantoio, Moraiolo, but not cultivars Rosciola and Piantone di Moiano. The Rugini Olive Medium including zeatin (4.5 μM) was used supplemented with a range of dikegulac concentrations (0–133.4 μM). An optimal result in number of shoots and nodes was obtained on cultivars Canino, Frantoio and Moraiolo at 66.7 μM dikegulac. Higher concentrations did not stimulate additional shoot and node formation and resulted in a drastic reduction in height of shoots. Elongated shoots were rooted and acclimatised and showed normal development compared to control plants.     

Cryopreservation of embryonic axes of trifoliate orange (Poncirus trifoliata [L.] RAF.)

Halved shoot bases of Allium tuberosum Rottl. ex Spreng. proliferated both axillary and adventitious shoots on B5 medium (1968) supplemented with either 6-benzylaminopurine (0.5 mg/l) or 1-naphthalene acetic acid (0.1 mg/l) and 2-isopentenyladenine (0.5 mg/l). In vitro shoots proliferated further numerous shoots upon subculture to fresh medium, and these shoots rooted spontaneously. Plantlets were transplanted successfully to soil and retained the diploid condition of the parents.Abbreviations B5 Gamborg et al. (1968) medium - BAP 6-benzylaminopurine - 2ip 2isopentenyladenine - NAA 1-naphthalene acetic acid     

Enhanced bud regeneration in aspen (Populus tremula L.) roots cultured in liquid media

 The regeneration potential of excised aspen (Populus tremula L.) roots cultivated in liquid medium, as affected by plant growth regulators and by the position of the isolated root explant on the main root, was investigated. The effect of various levels of benzyladenine (BA) and thidiazuron (TDZ) on bud regeneration in root explants was studied. TDZ in the medium had a marked effect on bud development as compared with BA, inducing a tenfold increase in the number of buds regenerated from various root explants. TDZ enhanced both root and root-borne shoot biomass production but reduced further shoot development and elongation. The position of the isolated root sections on the main root affected regeneration, the proximal sections further away from the root tip producing the highest number of buds per explant in both BA and TDZ treatments. Buds regenerated in close proximity to the site of lateral roots in BA-treated roots, while in TDZ-treated root sections, the buds formed all over the root regardless of the presence of lateral roots. The buds developed from inner cortical and sub-epidermal cell layers, disrupting the epidermis and the inner layers. Root biomass production and growth was greatly enhanced in well-aerated bioreactor culture in the presence of 4.5×10^–2 μM TDZ. A high number of the root-borne shoots could be rooted and converted to plantlets. However, while shoots regenerated in a medium with BA rooted well in a growth regulator-free medium, shoots formed in a medium with TDZ required auxin for rooting. Roots cultured in the presence of ancymidol, a gibberellin biosynthesis inhibitor, regenerated non-hyperhydric bud clusters and hyperhydric shoots. These were separated mechanically, subcultured to growth and rooting medium and transplanted ex vitro resulting in phenotypically true-to-type plantlets. The potential of liquid cultures for aspen shoot biomass production from roots is discussed. Received: 24 January 2000 / Revision received: 6 March 2000 / Accepted: 7 March 2000     

In vitro induction of multiple shoots and plant regeneration in cotton (Gossypium hirsutum L.)

Induction of multiple shoots in cotton (Gossypium hirsutum L. cv. Anjali-LRK 516) has been achieved with cotyledonary nodes devoid of cotyledons and apical meristems. Explants from 35-day-old seedlings yielded the maximum number of shoots (4.7 shoots/explant) using Murashige and Skoog (MS) basal medium supplemented with 6-benzylaminopurine and kinetin (2.5 mg/1 each). Explants from 35-day-old seedlings raised in glass bottles produced a higher number of multiple shoots (8.3 shoots/explant) than those grown in glass tubes and cultured on the same shoot induction medium. Elongation of multiple shoots was obtained on liquid or agar MS basal medium without phytohormones. In vitro shoots were rooted on half-strength agar-solidified MS basal medium or with 0.05 or 0.1 mg/1 naphthaleneacetic acid. Hardening and survival of tissue culture plantlets was 95% under greenhouse conditions.Abbreviations BAP 6-Benzylaminopurine - GA₃ Gibberellic acid - MS Murashige and Skoog medium - NAA -Napthaleneacetic acid     

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.     

Alterations in growth of tissue-cultured tansy (Tanacetum vulgare L.) treated with antibiotics

Effects of three antibiotics (cefotaxime, rifampicin and gentamicin) were tested on in vitro shoots cultures of tansy (Tanacetum vulgare L.)- These antibiotics were selected because endophytic bacteria isolated from the in vitro shoot cultures of tansy showed that all bacteria were Gram-negative and their growth was reduced by these three antibiotics. Five isolates were Enterobacteriaceae, three were fluorescent Pseudomonas, and two were aerobic bacteria. Increased concentrations of antibiotics caused usually linear or quadratic changes on the initiation of shoot growth, shoot number, growth rate, and shoot height. These changes and changes in pH of the culture media were tansy genotype-dependent following treatments with gentamicin. Also, the treatment with rifampicin or cefotaxime showed a genotype-dependent effect, because they resulted in significantly higher percentage of rooted plants in one of the three tansy genotypes tested. The growth rate and length of shoots were reduced in the media containing both gentamicin and rifampicin, but less so than in media containing both gentamicin and cefotaxime.     

Plant regeneration from cotyledonary node explants of mungbean (Vigna radiata (L.) Wilczek)

Sexually-mature mungbean (Vigna radiata (L.) Wilczek) plants were efficiently regenerated from cotyledonary node explants. The explants were capable of directly developing multiple shoots on basal media devoid of any growth regulators. The shoot multiplication was influenced by media composition, growth regulators, age of donor seedling and explant type. The explants with both the cotyledons attached to the embryonic axis excised from 4-d-old seedlings, produced the highest number of shoots (5 or 6) in 100% of the cultures within 2 weeks on B₅ basal medium (BBM) containing BAP or 2-iP, respectively, (at 5x10^–7M) and 3% sucrose. Shoots elongated and developed better using BAP. Increasing micronutrients, carbohydrate and nitrogen levels in the medium above the original formulation of B₅ basal medium appeared to be of no benefit for increasing the number of shoots. The shoots were rooted on basal MS medium or MS containing 10^–6 of NAA, IAA or IBA. This protocol was found applicable to six other cultivars of mungbean. One hundred rooted shoots were successfully established in soil in the glasshouse, where 90% of them survived. The regenerated plants flowered precociously, but produced normal pods and viable seeds.Abbreviations BAP 6-benzylaminopurine - KIN kinetin - 2-iP 6- — -dimethylallyl aminopurine - AdS adenine sulphate - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium - B₅ Gamborg et al. (1968) medium - C medium MS salts + B₅ vitamins     

Organogenesis and plant formation from cotyledon explant cultures of wild turnip rape (Brassica tournefortii L.)

Cotyledon explants of Brassica tournefortii L. were excised from germinated seedlings and cultured on Murashige & Skoog's [6] basal medium supplemented with various combinations of cytokinins and auxins, Both cytokinin and auxin were required for induction of shoot organogenesis. Of the three cytokinins tested (in combination with a low concentration of IAA), kinetin was found to be the best for shoot regeneration. On this medium, cotyledonary explants invariably underwent callusing followed by multiple shoot formation. NAA in combination with any of the three cytokinins yielded a reduced number of shoots or none, but favoured good callus growth. Callus so produced also regenerated shoots when subcultured on media containing high concentration of KIN or ZEA and low concentration of IAA. Shoots were rooted during prolonged incubation on the same medium or on MS medium free of growth regulators. Mature plants were grown in the greenhouse.