Influence of growth regulators on plant regeneration from epicotyl and hypocotyl cultures of two groundnut (Arachis hypogaea L.) cultivars

This study was designed to evaluate the effect of phytohormones on plant regeneration from epicotyl and hypocotyl explants of two groundnut (Arachis hypogaea) cultivars. Explants cultured on media with auxins and in combination with cytokinin produced high frequency of callus. After four weeks, callus from these cultures was transferred to medium with cytokinin and reduced auxin, shoot buds regenerated from the cultures. A high rate of shoot bud regeneration was observed on medium supplemented with 2.0 mg/L BAP and 0.5 mg/L NAA. Among the different auxins tested, NAA was found to be most effective, producing the highest frequency of shoot buds per responding cultures. Of the two explants tested, epicotyl was found to be best for high frequency shoot bud regeneration. Multiple shoots arose on MS medium supplemented with BAP or kinetin (1.0–5.0 mg/L) plus IBA (1.0 mg/L), with maximum production occurring at 5.0 mg/L. The elongated shoots developed rootsin vitro upon transfer to MS medium supplemented with NAA or IBA (0.5–2.0 mg/L) and kinetin (0.5 mg/L) for 15 days.In vitro produced plantlets, were transferred to soil and placed in a glasshouse developed successfully, matured, and set seeds.     

Micropropagation of Sesbania aculeata (Pers.) by adventitious organogenesis

Multiple shoots differentiated from hypocotyl explants of Sesbania aculeata (Pers.) syn S. cannabina (Retz.) Pers., a leguminous woody shrub, when cultured on Murashige and Skoog's basal medium supplemented with auxin (IBA, NAA) or auxin and cytokinin (IBA + BAP, NAA + BAP). Shoot budding occurred directly from the explant as well as from callus. Differentiation of shoot and root occurred in one step in the same concentration of auxin or auxin and cytokinin. Elongation of shoots occurred in the shoot induction medium.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

Regeneration of the aquatic monocot Aponogeton madagascariensis (lace plant) through callus induction

The lace plant (Aponogeton madagascariensis) is an aquatic monocot native to Madagascar that forms perforated leaves via developmentally regulated programmed cell death (PCD). Although a technique for culturing the lace plant under axenic conditions through corm separation has been developed, it is less productive for mass propagation of this species. Thus, an alternative method was investigated using 27 plant growth regulator (PGR) combinations to induce callus and subsequent whole plant regeneration. Combinations of the auxin picloram and the cytokinin thidiazuron (TDZ) successfully induced callus in corm sections, but no shoot regeneration was observed. Successful results for both callus induction and shoot regeneration were achieved using immature inflorescences with the PGR combination of 2 mg/L of the cytokinin 6-benzyaminopurine (BAP) and 2 mg/L of the auxin 1-napthaleneacetic acid (NAA) under light conditions, resulting in the regeneration of over 70 plants. The protocol shows both reproducibility and consistency in results; therefore, it is concluded that a technique for mass propagation of the lace plant through callus has been established. This technique may be useful in the study of other aquatic plant species, as well as in the study of developmental PCD in the lace plant itself.     

A regeneration protocol for sunflower (Helianthus annuus L.) protoplasts

Summary Hypocotyl protoplasts of four different Helianthus annuus genotypes were cultivated for 22–28 days in agarose droplets covered with liquid medium. In the first week, supplementation of the medium with plant growth regulators was at a 0.8/1 ratio of cytokinin and auxin followed by a high auxin concentration in the second week and a cytokinin to auxin ratio of 8/1 in the third and fourth week. Following transfer onto solid medium containing cytokinin and auxin in a proportion of 40/1 morphogenic callus started to form globular structures that developed into leaf primordia. Subsequent shoot elongation and rooting were obtained on hormone free medium after dipping the cut shoots into high auxin solution. Thirteen weeks after protoplast isolation, plantlets could be transferred to the greenhouse. Shoot regeneration was obtained for all four cultivars (Florom-328, Cerflor, Euroflor, Frankasol) at different rates reflecting their regenerative potential.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FeNaEDTA ethylenediamine tetraacetic acid ferric sodium salt - IAA indole acetic acid - MES morpholinoethane sulfonic acid - NAA 1-naphtalene acetic acid     

Combination of 6-Benzylaminopurine and Thidiazuron Promotes Highly Efficient Shoot Regeneration from Cotyledonary Node of Mature Peanut (<i>Arachis hypogaea</i> L.) Cultivars

Efficient in vitro plantlet regeneration is an important step to successfully transform genes for the improvement of agronomic traits. A combination of 6-benzylaminopurine (BAP) and thidiazuron (TDZ) plant growth regulators was applied to evaluate shoot regeneration capacity whereas α-naphthalene acetic acid (NAA) combination with 6-benzylaminopurine (BAP), and 2, 4-dichlorophenoxyacetic acid (2, 4-D) with 6-benzylaminopurine were tested to optimize root induction for two peanut cultivars. The result showed combination (BAP with TDZ) was found to be effective in promoting shoot. The highest shoot regeneration frequency (93%) was obtained on a medium supplemented with 4 mg/L BAP and 0.5 mg/L TDZ while an average regeneration frequency (87%) was achieved in a medium containing combinations of 2 mg/L BAP with 1 mg/L TDZ. The shooting rate increased for both cultivars as the concentrations of BAP increased and TDZ decreased. The highest rooting rate (93%) was obtained on a medium supplemented with 3.5 mg/L NAA with 2.5 mg/L BAP for both cultivars. The rooting rate increased as the concentration of auxin to cytokinin ratio increased. The maximum rooting rate (83%) was obtained on MS medium supplemented with 0.3 mg/L 2, 4-D with 0.2 mg/L BAP for the cultivar N3. The result indicated that BAP with NAA was much better than BAP with 2, 4-D in rooting rate. Thus, the protocol developed was genotype independent and effective for peanut tissue culture.     

In Vitro Regeneration from Seedling Organs of Brassica oleracea var. italica Plenck cv. Green Comet I. Effect of Plant Growth Regulators

The calabrese cultivar Brassica oleracea var. italica cv. GreenComet was used in a study of the effects of exogenous hormoneson the growth and differentiation of seedling organs in vitro.Four types of explants were tested: hypocotyl segments, rootsegments, primary leaf discs and cotyledon discs. These explantswere incubated on media containing factorial combinations ofBAP x IBA, BAP x NAA, KN x IBA and KN x NAA (all at 0, 0.1,10 and 10.0mg l^–1). Hypocotyls were the most regenerativeexplants; shoot production was favoured by cytokinin: auxinratios greater than one and was decreased by IBA at 10 mg l^–1when callus was produced. Shoot formation from root explantsoccurred either in the absence of hormones or with low concentrations;no shoot was produced when any hormone was present at 10 mgl^–1. In contrast, shoot production from primary leaf diseswas favoured by high concentrations of both auxin and cytokininwith the combination of BAP and IBA the most effective. Shootproduction from cotyledon discs was sporadic with no consistentresponse on any auxin/cytokinin combination. After further experimentson the optimization of hormone concentration, the followingcombinations were chosen as allowing reliable regeneration:0.1 mg l^–1 BAP+0.1mg l^–1 IBA for hypocotyl segments,0.075 mg l^–1 KN +0.025 mg l^–1 IBA for root segments,and 5.0 mg l^–1 BAP+5.0 mg l^–1 IBA for leaf discs. Brassica oleracea var. italica, calabrese, tissue culture, seedling, auxin, cytokinin     

Expression of a KDEL-tagged dengue virus protein in cell suspension cultures of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> and <Emphasis Type="Italic">Morinda citrifolia</Emphasis>

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.     

Plant regeneration from petal explants of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.     

Induction of multiple shoots by thidiazuron from caryopsis cultures of minor millet (Paspalum scrobiculatum L.) and its effect on the regeneration of embryogenic callus cultures

Caryopsis culture of a minor millet (Paspalum scrobiculatum L. cv. PSC 1) on N₆ medium supplemented with high concentrations of thidiazuron (TDZ, 11.25 µM and 22.5 µM), a phenylurea derivative known to simulate cytokinin action, resulted in the formation of multiple shoots from the base of the seedling. This is the first time that multiple-shoot formation by a seedling cultured on TDZ without a callus interphase has been reported in graminaceous crop plants. The presence of a cytokinin, 6-benzylaminopurine (BAP), at low or high concentrations failed to evoke any morphogenic response. The presence of the auxin 2,4-dichlorophenoxyacetic acid (2,4-D, 4.5 µM) either alone or with BAP (4.5 µM) resulted in the formation of embryogenic callus from the base of the seedlings, which subsequently differentiated into somatic embryos. The combination of TDZ and the auxin (4.5 µM, 2,4-D) in the medium stimulated the differentiation of shoot buds in embryogenic callus cultures. This effect of TDZ, noted for the first time in a monocotyledonous plant, was evident in terms of a significant increase in the frequency of shoot-bud formation in embryogenic callus cultures and occurred only at a high concentration of TDZ (11.25 µM). This requirement for a high concentration of TDZ for the induction of multiple shoots from cultured seedlings or shoot buds in an embryogenic callus culture of a monocot is contrary to its effect at low concentrations in dicotyledonous plants. Complete plantlets, derived either from somatic embryos or shoot buds, could be regenerated on hormone-free basal medium or on basal medium fortified with activated charcoal (0.5%). Following a gradual acclimatization in a culture room, these regenerants survived on transfer to soil and ultimately set seed.     

Histological study of embryo-like structures initiated from hypocotyl segments of flax (Linum usitatissimum L.)

Cultivation of flax hypocotyl segments on MS medium supplemented with auxin (2,4-d, NAA) and combination of auxin (NAA) and cytokinin (BAP, zeatin) resulted in production of callus on the cut ends of segments and prolonged cultivation in globular structures resembling early stages of somatic embryos. Embryo-like structures protruded on the surface directly from the subepidermal layers of hypocotyl segments. Despite these globular structures closely resembling somatic embryos, histological observations did not reveal their embryogenic character–organogenesis was the predominant developmental morphogenic pathway. Based on our experiments, as well as on critical revision of existing reports on flax somatic embryogenesis, we conclude, that there has not yet been convincing histological proof of somatic embyogenesis from flax hypocotyl segments.     

In vitro regeneration of Echinacea purpurea from leaf explants

Efficient plant regeneration was achieved via organogenesis from callus cultures derived from leaf tissue of Echinacea purpurea. Proliferating shoot cultures were obtained by placing leaf explants on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) combinations. MS medium supplemented with BAP (4.44 M) and NAA (0.054 M) was the most effective, providing high shoot regeneration frequencies (100%) associated with a high number of shoots per explant (7.7 shoots/explant). Plantlets were rooted on MS medium alone or in combination with different concentrations of indole-3-butyric acid (IBA), and high rooting and survival was achieved using MS media without plant growth regulators (PGR). All plantlets survived acclimatization producing healthy plants in the greenhouse. This study demonstrated that adventitious shoot regeneration of E. purpurea from leaf explants can be a useful method for the multiplication of this important medicinal plant.     

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     

Highly Efficient and Rapid Plant Regeneration in Citrus sinensis

Sweet orange (Citrus sinensis L Osbeck, var Nagpur) was explored for efficient multiple shoot regeneration and rooting in different media. The influence of phytohormones and carbon source on the in vitro morphogenesis of sweet orange epicotyl explants was investigated. Among the various concentrations and combinations of auxins (IAA and NAA) and cytokinins (BAP, Kn, Zn, and TDZ) tried, MT (Murashige and Tucker) medium fortified with benzylaminopurine (BAP) at 1 mg l^?1 without auxin had a strong promotive effect on shoot regeneration, and elucidated best morphogenic response from one-month-old etiolated epicotyl explants. A 100% regeneration frequency was obtained, and multiple shoots with an average of 8.24 shoots per explant were produced on all of the explants. Root formation was seen in response to all the three auxins viz. IBA, NAA and IAA, but the best response with rapid induction was observed under the influence of indole butyric acid (IBA) at 1 mg l^?1. Sucrose was observed to be at par with maltose as carbon source to support shoot regeneration. This study provided promising results, holds potential to be routinely employed for in vitro regeneration of important cultivars of Citrus spp, and can be incorporated for genetic transformation studies in citrus.     

Large scale micropropagation ofQuercus robur L. using adenine-type cytokinins and thidiazuron to stimulate shoot proliferation

High rates of oak shoot multiplication were achieved on low salt nutrient media (BTM, WPM) supplemented with adenine type cytokinin or BAP. Low concentration of (0.2-0.6 mg 1^-1) stimulated formation of a large number of axillary shoots and their elongation. Thidiazuron in very low concentration (0.001 - 0.002 mg 1^-1) promoted shoot proliferation, in high concentration stimulated formation of large callus. More than twelve thousand micropropagated shoots were rooted in low salt agar media supplemented with low concentration of auxin (IBA or NAA 0.2 - 0.5 mg 1^-1). High rooting percentages (81 %) were obtained. Survival of the mieropropagated plantlets transplanted into soil was high (78 %). Micropropagated trees planted in the field withstood severe winter frosts without significant losses. At the end of the fifth growing season trees attained considerable size.     

Effect of genotype and medium composition on linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis>) ovary culture

Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders to develop new cultivars in a shorter time period. Many research groups successfully created new linseed genotypes through anther culture; however ovary culture has been the subject of only a few earlier studies. In the present study, the effect of genotype and growth regulators combination on callus induction and shoots regeneration in ovary culture of nine commercially important linseed cultivars was investigated. Ovaries were cultured on modified MS medium supplemented with three different combinations of plant growth regulators. Variable callogenic responses were expressed by all of the genotypes tested on different induction media. The results suggested that specific combination of growth regulators for callus induction must be designed for each genotype. Shoot regeneration from ovary derived callus is a critical phase of the whole gynogenetic process. Differences in adventitious shoot formation frequency among genotypes were demonstrated and four responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’ manifested the highest adventitious shoot formation frequency with a high number of shoots per explant. The optimum ratio of growth regulators for shoot regeneration was shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaphir’ showed the highest shoot regeneration frequency when callus had originated on induction medium supplemented with 2 mg L⁻¹ BAP and 2 mg L⁻¹ NAA, while combination of 1 mg L⁻¹ BAP and 2 mg L⁻¹ IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest rate of shoots per explant has been obtained in second subculture.     

Micropropagation ofLarix decidua Mill. andpinus sylvestris L.

Shoot multiplication of Larixdecidua was achieved using axillary and adventitious buds. The formation of axillary buds was stimulated on shoot tips soaked in a cytokinin solution (BAP 10-50 mg 1⁻¹ for 2–4 h. Adventitious buds were induced on cotyledons, needles and vegetative buds cultured on WPM or QL medium supplemented with cytokinin (BAP 1–3 mg 1⁻¹). The shoot formation from induced axillary and adventitious buds was promoted on WPM or QL medium containing a low concentration of auxin (IBA 0.1 mg 1⁻¹). Shoot multiplication of Pinussylvestris was stimulated on WPM, MS, and QL media supplemented with a low concentration of cytokinin (BAP 0.2 mg 1⁻¹) and auxin (IBA 0.1 mg 1⁻¹). Shoot segments produced 2–5 new axillary shoots within 4–5 weeks. Root initiation was stimulated on larch and pine shoots cultured first on WPM supplemented with auxins (NAA and IBA) and later transferred to auxin-free medium.     

In vitro culture of petioles and intact leaves of sugar beet (Beta vulgaris)

Methods are described for obtaining explants which produce adventitious shoots, for subsequent stimulation of rooting and then transplanting using six commercial sugar-beet cultivars. The rate of adventitious shoot regeneration from petioles or intact leaf explants was affected by the source of donor plants, cytokinin type (BAP or Kin) and concentration and cultivar. Increasing the sucrose concentration of the medium from 3% to 5% or 8% had no apparent effect. Adventitious shoots could be produced directly from callus formed on the base of the petioles. In general adventitious shoots were produced on either the concave surface of the petiole or from the callus, occasionally simultaneously on both, and on the convex surface of the petiole in intact leaf explants. The highest rooting rate with 3% sucrose and 1.0 mg l^–1 NAA was obtained using half-strength MS medium. There was considerable variation in the propagules from petioles or callus indicating that this system may provide valuable somaclonal variation.Abbreviations BAP benzylaminopurine - IBA indole-3-butyric acid - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid Author for correspondence     

Micropropagation of three berry fruit species using nodal segments from field-grown plants

Plant micropropagation of blueberry (Vaccinium corymbosum L. cv. Berkeley), blackberry (Rubus sp. cv. Smoothstem) and raspberry (Rubus idaeus L. cv. Gradina) was carried out from nodal segments of adult field‐grown plants. Hardwood and softwood cuttings were studied as explant sources. The cultures successfully established were softwood from all three species, and hardwood only from blueberry. Shoot‐bud establishment from blueberry was achieved by culturing explants in WPM salts with MS vitamins for 15 days, and then 30 days in the same medium with 18 mM Zeatin. The best results of multiplication were obtained in the same medium with 25 mM 2iP. For blackberry, shoot‐bud establishment was achieved by culturing explants in MS medium for 15 days, and then in the same medium with 4 mM BA and 0.25 mM IBA. This medium was also the best for blackberry multiplication. Raspberry explants (cvs Gradina and Willamette) were cultured in MS medium for 15 days and then transferred to MS medium supplemented with 4 μm BA and 0.25 mM IBA. After 30 days of culture, only ‘Gradina’ explants survived, from which shoot‐bud establishment was obtained in a modified MS medium (Anderson's macronutrients except calcium, with Sequestrene as the iron source) with the same growth regulators. Multiplication was achieved by subculturing explants in the same medium either with 4 mM BA plus 0.25 mM IBA or with 8 mM BA plus 0.25 mM IBA. Shoots of at least 1 cm in length from all species were rooted ex vitro in a mixture of peat and Perlite (1:1, v/v) in a mist chamber, and 100% of rooting plants were acclimated. Bacterial, fungal and viral diseases were detected in stock plants, while tests carried out in both shoots and regenerated plants revealed the absence of any kind of disease.     

High-frequency callus induction and plant regeneration in Tripsacum dactyloides (L.)

Summary A protocol for high-frequency callus, somatic embryogenesis, and plant regeneration for Tripsacum is described. Plants were regenerated from complete shoot meristems (3–4 mm) via organogenesis and embryogenesis. In organogenesis, the shoot meristems were cultured directly on a high cytokinin medium comprising 5–10 mgl⁻¹ (22.2–44.4 μM) 6-benzyladenine (BA). The number of multiple shoots varied from six to eight from each meristem. The time required for production of plants from organogenesis was rapid (4–6 wk). In contrast, callus was induced on an auxin medium and continuously cultured on an auxin medium for production of somatic embryos. Prolific callus with numerous somatic embryos developed within 3–4 wk when cultured on an auxin medium containing 5 mgl⁻¹ (22.6μM), 2,4-dichlorophenoxyacetic acid (2,4-D). The number of shoots induced varied from two to five per callus. Regardless of the cultivars used, the frequency of callus induction and plant regeneration was between 48% and 94%. The seed germination procedures also were modified and resulted in a maximum of 60–80% seed germination. Finally, the rate of T-DNA transfer to complete shoot meristems of Tripsacum was high on the auxin medium and was independent of whether super-virulent strains of Agrobacterium were used or not.