Regeneration in cultures of Papaver bracteatum as influenced by growth hormones and temperature

Callus was induced on Papaver bracteatum Lindl. seedlings inoculated on Murashige & Skoog (1962) medium supplemented with -naphthaleneacetic acid (NAA) (1.0 mg 1^-1) and benzylamine purine (BA) (0.5 mg 1^-1). Subculture resulted in excellent callus proliferation but no organogenesis. Shoots were regenerated in cultures grown on MS medium containing NAA (1.0 mg 1^–1), BA (0.5 mg 1^–1) and casein hyrdrolyzate (2.0 mg 1^-1). The shoots developed into plantlets after 8 weeks of culture, and were induced to root on 1/2 MS without the addition of growth regulators. The rooted plantlets were transferred to soil after hardening.MS at full strength was found inhibitory for callus induction and proliferation, but 1/2 MS was suitable. Similarly callus growth was very slow at 25°C but increased when the temperature was lowered to 15°C as did bud initiation.Abbreviations BA benzylamine purine - IAA indoleacetic acid - K kinetin (6-furfurylamino purine) - NAA -naphthalene acetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Induction of somatic embryogenesis and organogenesis in <Emphasis Type="Italic">Oldenlandia umbellata</Emphasis> L., a dye-yielding medicinal plant

Oldenlandia umbellata L., commonly known as “Indian madder”, is an ancient Indian herb valued as a source of red color dye and various medicinal products. In this study, successful protocols have been developed for induction of somatic embryogenesis and organogenesis in O. umbellata. Emerging young leaves, shoot apices, and stems were used as explants, grown on Murashige and Skoog (MS) media supplemented with various auxins, including indole acetic acid, indole butyric acid, napthaleneacetic acid (NAA), and 2,4-Dichlorophenoxyacetic acid, each at levels ranging between 0.1 and 0.5 mg/l, cytokinins, including benzyladenine (BA) and kinetin, each at concentration ranging between 0.5 and 5 mg/l, with and without coconut milk (CM) at levels of 0.5–5%. For callus induction, NAA at 2.5 mg/l was optimal; while, for rapid embryogenic callus induction, 0.2 mg/l NAA, 0.5 mg/l BA, and 0.1% CM induced the highest frequency (95.86%). Shoots developed upon transfer of embryogenic calli to MS medium containing 1.5 mg/l BA, 0.3 mg/l NAA and 1% CM. For root induction, 0.3 mg/l NAA and 1.0% CM promoted highest and earliest rooting. C. Rajasekaran contributed equally to this work.     

Phenylacetic acid induced organogenesis in cultured leaf segments of Dianthus chinensis

Summary Shoot regeneration was achieved from leaf derived callus of Dianthus chinensis using Phenylacetic acid (PAA). Callus from basal leaf segments, raised on Murashige and Skoog's (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or 1-Naphthaleneacetic acid (NAA) in combination with 6-benzylamino purine (BAP), was subcultured on medium supplemented with BAP in combination with 2,4-D, NAA or PAA. Shoots were induced only when leaf derived callus was subcultured on medium containing BAP (2.0, 5.0 mg/l) in combination with PAA (0.5, 1.0 mg/l). No shoot regeneration was observed when 2,4-D, NAA or BAP were used in the medium either singly or in different combinations. These results demonstrate that PAA in combination with BAP was essential to trigger shoot regeneration from cultured leaf callus of D. chinensis.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DPX dibutylphthalate xylol - MS Murashige and Skoog (1962) basal medium - NAA 1-Naphthaleneacetic acid - PAA Phenylacetic acid     

Micropropagation ofActinidia polygama from fruit galls

Callus was induced from the fruit galls of Actinidia polygama on the B5 medium supplemented with NAA, BA and kinetin (1 mg l⁻¹ each). When the callus was subcultured on the same medium 3 times, shoot primordia and adventitious roots appeared on the surface of the callus. Shoots subcultured on growth regulator free MS medium or supplemented with NAA (1 mg l⁻¹) formed roots. Chromosome numbers in the root tip of regenerated plantlets were determined as 2n=58, which was the same as that of the mother plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regeneration of Lycium barbarum L. plants from leaf tissue,callus culture and callus protoplasts

The possibility of plant regeneration from leaf tissue, callus and callus protoplasts of Lycium barbarum L. has been studied. Leaf segments were cultured on B5 medium (Gamborg et al. 1968) containing 1.5 mg/1 6-benzylaminopurine and 0.5 mg/1 -naphthaleneacetic acid. Regeneration of shoots was initiated after 30 days of cultivation. Callus was obtained from leaf and internode tissues on MS medium (Murashige and Skoog 1962) containing 0.4 mg/1 of 2,4dichlorophenoxyacetic acid. Subsequently, callus was successfully subcultured on the same medium with 1 mg/l of 2,4-dichlorophenoxyacetic acid and 0.2 mg/l -naphthaleneacetic acid. Organogenesis in callus culture was obtained in the course of 40 days after transferring to TM-4 (Shahin 1984). Protoplasts were isolated from callus tissue grown in vitro using an enzymatic method. Cell colonies, minicallus formation and organogenesis were obtained. Shoots were rooted on Murashige and Skoog medium containing 0..1 mg/l -naphthaleneacetic acid. Regenerated plants were transferred to soil and were grown to maturity. Regenerated plants carried normal morphological traits.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - Zea zeatin - GA₃ gibberellic acid - MS Murashige and Skoog medium - B5 Gamborg medium     

Production of solasodine by Solanum laciniatum using plant tissue culture technique

Leaf and hypocotyl explants of 15 days old aseptically grown seedlings of Solanum laciniatum were cultured on MS medium supplemented with NAA (2 mg/l) and kinetin (0.5 mg/l) for callus initiation. For maintenance and proliferation of callus MS medium supplemented with 2,4-D (1 mg/l) and kinetin (0.5 mg/l) was used. The growth of the calli derived from hypocotyls increased with time of incubation and remained almost constant after 45 days. The solasodine content in callus culture was maximum after 30 days of incubation. Addition of L-arginine in the medium (50-150 mg/l) increased growth as well as chlorophyll content in the callus culture. The solasodine content also increased up to 1.2 to 1.4 times in these cultures. High frequency shoot regeneration was obtained in MS medium having BA (4 mg/l) and IBA (0.25 mg/l). For shoot multiplication, MS medium having BA (4 mg/l) was used. Shoots rooted on the same medium. Organogenesis promoted solasodine accumulation in the cultures. Regenerated shoots yielded higher solasodine content than undifferentiated as well as organogenic callus. Solasodine contents in the regenerated shoots was found to be 10 times higher than the callus culture and approached towards the field grown plants. Thin layer chromatography revealed the presence of three compounds. The most predominant spot (Rf 0.789) corresponded to the reference solasodine.     

Regeneration of plantlets from the callus of stem segments of adult plants of Ficus religiosa L.

Stem segments of adult plants of Ficus religiosa L. cultured on MS medium containing 1.0 mg/l 2,4-D produced callus. Shoots were regenerated when the induced calli were transferred to medium supplemented with 0.05 to 2.0 mg/l BAP. Callus derived shoots produced roots and developed into plantlets when transferred to medium supplemented with 1.0 mg/l NAA.Abbreviations MS Murashige and Skoog (1962) - BAP 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Tissue Cultures of Phaseolus vulgaris L.

Abstract

Callus production and plant regeneration from different explants of Phaseolus vulgaris L. cv. Giza are reported. Calli cultures were induced from leaf, hypocotyl, embryo and root explants. Rapid growth of callus was achieved by leaf explants cultured on MS salts, B5 vitamins and supplemented with 2,4— dichlorophenoxyacetic acid (2, 4—D)+0.5 mg/l kinetin (kin). Addition of casein hydrolysate at 2 g/l to maintenance medium enhanced callus growth and hindered the early appearance of necrotic parts. This report also provides a detailed method for production of multiple shoots directly from the wounded edges of immature cotyledon explant via organogenesis on 1 mg/l benzyladenine (BA) or indirectly on 0.5 mg/l naphthaleneacetic acid (NAA)+2 mg/l BA. The regeneration of bean plants through the two ways described here (direct or indirect) may be of use in genetic improvement of bean.     

Plant regeneration from leaf base callus of turmeric and random amplified polymorphic DNA analysis of regenerated plants

Callus cultures were initiated from leaf bases of turmeric on Murashige and Skoog's basal medium (MS) supplemented with dicamba, picloram (2 mg l⁻¹) or 1-naphthaleneacetic acid (NAA) (5 mg l⁻¹) in combination with benzyladenine (BA) (0.5 mg l⁻¹). On transfer of callus cultures to medium supplemented with benzyladenine (BA) (5 mg l⁻¹) in combination with triiodebenzoic acid (TIBA) or 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1 mg l⁻¹), green shoot primordia were seen to differentiate from the surface of the callus. On transfer of regenerating cultures to half MS media supplemented with Kn, shoot primordia developed into well developed shoots. When shoots were transferred to medium devoid of phytohormones, complete rooted plants were obtained. Ninety percent of the plants survived to maturity on transfer to soil. Random Amplified Polymorphic DNA (RAPD) analysis of eight regenerated plants using 14 primers when separated on non-denaturing polyacrylamide gels showed 38 novel bands. About 51 bands present in the control were absent in the regenerants. The result indicates that variation at DNA level has occurred during in vitro culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration through callus cultures derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.)

Efficient plant regeneration was achieved from callus derived from immature-cotyledon explants of oleaster (Elaeagnus angustifolia L.). Calli were obtained on MS media containing 3% sucrose and different concentrations of TDZ. The highest rate of green, compact and nodular callus was formed on MS medium supplemented with 1 mg/l of TDZ. Shoot organogenesis was achieved when the callus was transferred onto MS media containing 3% sucrose and BA alone (05–4 mg/l) or BA (0.5 and 1 mg/l) combined with NAA or IAA (0.5 and 1 mg/l). Maximum organogenesis was obtained with 1 mg/l BA in combination with 0.5 mg/l NAA. Rooting of the shoots was achieved on MS medium supplemented with 0.2 mg/l IBA. Regenerated plantlets were acclimatized and successfully transplanted to soil.     

Shoot regeneration from stem and leaf callus of Eucalyptus tereticornis

Adventitious shoots were obtained from leaf and stem callus of Eucalyptus tereticornis SM. Callus was induced on B5 medium with 0.1 mg/l benzyladenine (BA) and 3 or 5 mg/l naphthalene acetic acid in the dark. Shoot initiation occurred on modified Woody Plant medium (mWP) containing 0.5 mg/l BA, 500 mg/l polyvinylpyrrolidone and 10% (v/v) coconut milk. Multiple shoots were also regenerated directly from hypocotyl segments of 4 to 6 week old seedlings on B5 medium with 0.5 mg/l BA. Regenerated shoots could be rooted with 100% efficiency on mWP medium containing 0.5 mg/l indolebutyric acid and transferred to soil in the greenhouse. Suspension cultures were obtained from the callus using B5 medium with 0.5 mg/l 2,4-dichlorophenoxyacetic acid. Callus clumps grew from less than 1 mm to 4–6 mm in diameter within two weeks on transfer to shoot regeneration medium but failed to form shoots or somatic embryos.Abbreviations BA Benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - PVP Polyvinylpyrrolidone - mWP modified Woody Plant medium Scientific Contribution No. 1689 from New Hampshire Agricultural Experiment Station.     

Optimisation of plantlet regeneration from leaf and nodal derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andrews

An indirect in vitro plant regeneration protocol for Vanilla planifolia has been established. Juvenile leaf and nodal segments from V. planifolia were used as explants to initiate callus. Nodal explants showed better callus initiation than juvenile leaf explants, with 35.0% of explants forming callus when cultured on Murashige and Skoog (MS) basal medium supplemented with 2.0 mg/l 1-naphthylacetic acid (NAA) and 1.0 mg/l 6-benzyladenine (BA). Almost 10.0% of juvenile leaf explants were induced to form callus on the MS basal medium containing 2.0 mg/l NAA and 2.0 mg/l BA, whereas no callus formed in the presence of any concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and BA. After 8 weeks, callus generated was transferred to MS basal medium containing 1.0 mg/l BA and 0.5 mg/l NAA. A mean number of 4.2 shoots per callus was produced on this medium, with a mean length of 3.8 cm after 8 weeks of culture. Roots formed on 88.3% of plantlets when they were cultured on MS medium supplemented with 1.0 mg/l NAA, with a mean length of 4.4 cm after 4 weeks of culture. Of the rooted plantlets, 90.0% survived acclimatisation and were making new growth after 4 weeks.     

In vitro studies on Pea (Pisum sativum L.): I. Callus formation,regeneration and rooting

Abstract

Callus production, shoot formation via organogenesis and rooting of the regenerated shoots are reported in an Egyptian variety of Pisum sativum L. Calli were initiated from hypocotyl, leaf, root and mature embryo explants when cultured on MS medium containing B5 vitamins and supplemented with 2 mg/l 2,4-D+1 mg/l kin. Among the different types of explants, hypocotyl showed best potential for callus proliferation. Hypocotyl, leaf and immature cotyledon explants were used for shoot organogenesis. The best results of shoot formation were achieved when hypocotyl explants were cultured on MS-medium supplemented with 2 mg/l BA+1 mg/l NAA. However, immature cotyledon explants showed the highest frequency of shoot formation with 1 mg/l BA. Data of in vitro rooting showed that maximum root frequency occurred on culture medium containing half strength of MS salts, 40 g/l sucrose and 2 mg/l NAA.     

Regeneration of garlic plants (allium sativum L., CV. “Chonan”) via cell culture in liquid medium

Summary Aiming at the genetic improvement of garlic cultivars, a cell suspension protocol was established which includes the induction of friable callus, establishment of cells in liquid medium, plating, regeneration, and bulb formation. Calluses of various textures from compact to friable and from green to yellowish were obtained by culturing explants excised from inner leaves of garlic bulbs on Marashig-Shoog (MS) medium with 2,4 dichlorophenoxy acetic acid (2,4-D), (1.1 mg/liter [5.0 μM]), picloram (1.2 mg/liter [5.0 μM]), and kinetin (2.1 mg/liter [10 μM]). Friable callus occurred on MS-A contained 2,4-D alone (1.0 mg/liter [4.52 μM]) and this callus was used to develop cell suspension cultures, which were maintained in liquid MS-B medium with a 2,4-D/benzyl adenine (BA) (0.5 mg/liter [2.25 μM]: 0.5 mg/liter [2.22 μM]) ratio. High plating efficiency was obtained on MS-C medium with different naphthalene acetic acid/BA combinations. Regeneration occurred after transfer of the caulogenic mass to MS-C medium containing 10 mg/liter (74.02 μM) and 20 mg/liter (148.04 μM) adenine for 60 days, followed by transfer to adenine-free medium. Plantlets transplanted to soil showed normal phenology. Shoots grown on modified MS medium supplemented with indolylbutryic acid (3.0 mg/liter [14.7 μM]) stimulated bulb formation by 30 days in culture.     

Influence of isolation technique of half-anthers and of initiation culture medium on callus induction and regeneration in Anthurium andreanum

Optimization of the isolation technique and initiation culture medium are two critical aspects that can determine the success of anthurium half-anther culture. Both aspects in half-anther culture of Anthurium andreanum Linden ex André cv. ??Tropical?? were studied and successfully improved. Untreated half-anthers, when cultured abaxial side down on medium, was the most suitable means of inducing callus. Callus formation was further improved by culturing half-anthers adaxial side down on Winarto-Teixeira (WT) medium (Winarto et al. in Plant Growth Regul 65:513?C529, 2011b) supplemented with 0.01?mg/l ??-naphthaleneacetic acid (NAA), 1.0?mg/l 6-benzyladenine (BA) and 0.5?mg/l thidiazuron (TDZ). Gelrite enhanced callus formation (compared to agar) when the concentration was reduced from 2.0 to 1.5?g/l on WT medium. Application of 0.5?mg/l 2,4-dichlorophenoxy acetic acid (2,4-D) in WT medium increased callus formation. Most callus formed when half-anthers were cultured adaxial-side down on WT medium supplemented with 0.5?mg/l 2,4-D in combination with 0.01?mg/l NAA, 1.0?mg/l BA and 0.5?mg/l TDZ using 1.5?g/l Gelrite. This ideal medium induced the growth of half-anthers, with as much as 38?% of half-anthers producing callus, or, on average, 3.4 half-anthers/treatment. Callus derived from this optimized protocol regenerated easily and could be multiplied on New Winarto-Teixeira medium (NWT) (Winarto et al. in Plant Growth Regul 65:513?C529, 2011b) containing 0.25?mg/l 2,4-D, 0.02?mg/l NAA, 0.75?mg/l BA, 1.5?mg/l TDZ and 2.0?g/l Gelrite. Shoots rooted well on hormone-free NWT medium with 2.0?g/l Gelrite. The plantlets could be easily acclimatized in a substrate of raw rice husk, burned-rice husk and organic manure (1:1:1, v/v/v) with a high survival (100?%) ex vitro in a greenhouse. The results of this study would benefit half-anther culture of other Anthurium cultivars, particularly at the initial stage of callus induction.     

High efficiency plant regeneration from petiole explants of Carica papaya L. through organogenesis

Callus cultures were obrained from petiole explants of Carica papaya on MS medium containing 0.5–10.5 μM α-naphthaleneacetic acid (NAA) in combination with 0.5–5 μM benzyladenine (BA). Hard-green calli were transferred to MS medium containing 100 mgl⁻¹ casein hydrolysate (CH) with specific BA-NAA formulation, where they developed adventitious buds within 2 weeks of culture. Maximum number of adventitious buds were obtained in 2 μM BA and 0.1 μM NAA. Shoot regeneration occurred from these adventitious buds by the end of the 4th week. Regenerated shoots were elongated in hormone-free medium and rooted in half-strength MS fortified with 3 UM NAA and 0.5 μM gibberellic acid (GA₃). The regenerants were transferred to soil after acclimatization.     

Aluminium-induced morphogenic and biochemical variations ofBacopa Monniera

Shoots and roots ofBacopa monniera (L.) Wettst. have been regenerated from nodal segments on MS medium containing combinations of NAA and BAP. The cultures showed 100% regeneration on MS (sucrose 2%) medium added with NAA (0.2 mg L^-1), BAP (0.5 mg L^-1) and glutamine (50 mg L^-1). Supplemented with aluminium chloride (up to 400 μM), this medium could ensure successful survival of regenerants. AH the regenerants, maintained on AlCl₃-supplemented medium for the last three years, failed to grow when transferred to AlCl₃-free media. Aluminium stress also induced synthesis of proline and proteins. The rate of photosynthesis decreased at increased aluminium concentrations.     

Propagation of the tropical tree,Leucaena leucocephala K67, by in vitro bud culture

A tissue culture method is described for clonal multiplication of Leucaena leucocephala K67 using single lateral bud explants from 2–3 m tall greenhouse grown trees. N-6 benzyladenine (BA: 3.0 mg.1^-1) and napthaleneacetic acid (NAA: 0.05 mg.1^-1) in Murashige & Skoog's (MS) medium were found to be best suited for multiple shoot differentiation in 4–5 week old cultures. Analysis of variance of the main treatment effects of BA and NAA on shoot parameters showed that BA significantly (P=0.001) affected shoot development while NAA did not. A shoot multiplication rate of 22±3.63 shoots per bud explant was obtained in 150 days on 1/2 strength MS medium with 3.0 mg.1^-1 BA and 0.05 mg.1^-1 NAA. Shoots developed adventitious roots within 15 days in 1/2 strength MS medium containing indole-3-butyric acid (IBA: 3.0 mg.1^-1) and Kinetin (0.05 mg.1^-1). Eighty percent of the transplanted plantlets are being grown in greenhouse conditions.     

Plant regeneration in Arachis pintoi (Leguminosae) through leaf culture

Plant regeneration in Arachis pintoi was obtained via two developmental pathways: organogenesis and somatic embryogenesis. Organogenic callus cultures were initiated from pieces of leaf on MS medium supplemented with NAA or 2,4-D in combination with BA, KIN or 2iP. The most suitable combination for plant regeneration through organogenesis was an initial medium composed of 10 mg/l NAA+1 mg/l BA followed by transfer of the callus to a shoot induction medium (MS+1 mg/l BA). Rooting of regenerated shoots was readily achieved by culture on MS+0.01 mg/l NAA. Embryogenic callus cultures were initiated from pieces of leaf on MS medium supplemented with PICL in combination with KIN, ZEA, BA or 2iP, and the most suitable combinations were 20 mg/l PICL+1 mg/l BA or 2iP. When pieces of embryogenic callus were subcultured on MS+1 mg/l BA, somatic embryos were differentiated and developed further into well-developed plants in MS+1 g/l AC followed by MS medium devoid of plant growth regulators. Received: 29 April 1999 / Revision received: 24 November 1999 / Accepted: 18 December 1999     

Callus induction and plantlet regeneration in Bixa oreliana L., an annatto-yielding tree

Summary A protocol has been developed for plantlet regeneration from seed callus of Bixa orellana L. Seeds demonstrated a high percentage of callus induction (63±7.3%) and a high yield (356±14.7 mg per seed) of white friable callus on Murashige and Skoog (MS) medium containing 5.0 μM l-naphthaleneacetic acid (NAA) and 2.5μM N ⁶-benzyladenine (BA) within 6 wk of culture in the dark. Callus induction frequency was greater under 24h dark as compared to 16h light/8h dark photoperiod or 24h light photoperiod. Increased myo-inositol (MI: 200mgl⁻¹) and addition of ascorbic acid (AA: 200 mgl⁻¹) to the culture medium positively improved callus induction frequency and growth. Shoot differentiation from white friable seed callus was best using 10.0 μM BA and 5.0 μM NAA, where the highest percentage of calluses forming shools (74.9±4.8%), the highest number of shoots per callus (six or seven) and the highest shoot-forming index (5.0) were obtained within 6 wk. Shoots elongated to 4 cm within 4 wk of transfer onto MS medium devoid of growth regulators. Shoots were rooted using half-strength MS medium containing 5.0 μM indole-3-butyric acid (IBA). About 85% of these plants were established in pots containing pure garden soil and organic manure after 3 wk of hardening. Regenerated plants were morphologically uniform with normal leaf, shape and growth patterns. These plants are currently being screened for the presence of agronomically useful genetic variants.