Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoog's basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l^-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l^-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.Abbreviations BAP 6-benzylamino purine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Enhancement of shoot regeneration potential by liquid medium culture from mature cotyledons of sunflower (Helianthus annuus L.)

We describe here a liquid culture system for the regeneration of shoots at high frequencies from mature cotyledon tissues of three genotypes of sunflower (Helianthus annuus L.) one of which had previously been found to be recalcitrant to regeneration when cotyledons were cultured on solid medium. Cotyledons were excised from 2-day-old seedlings and incubated in liquid Murashige and Skoog's modified medium supplemented with 5.4 M naphthaleneacetic acid (NAA) and 4.4 M benzylaminopurine (BAP). After two weeks in culture, the whole upper surface of regenerating explants was covered with green shootlets. The percentages of regenerating explants of three genotypes varied between 60 and 70%, and the number of shoots per regenerating explant was highly increased. The shootlets were transferred to solid Murashige and Skoog's medium allowing shoot development, then to rooting medium. Rooted plantlets were successfully acclimatized and gave fertile plants. The role of liquid medium culture in the induction of sunflower regeneration is discussed.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Plant regeneration from callus cultures of Valeriana wallichii DC.

Petiole expiants of Valeriana wallichii. DC., a threatened medicinal plant, were used for inducing callus. Optimum callus formation was observed on Murashige and Skoogs' (1962) medium supplemented with 3.0 mg/l NAA and 0.25 mg/l Kn. Shoot regeneration was achieved upon transferring the callus to medium containing 1.0 mg/l Kn and 0.25 mg/l NAA. Complete plantlets were obtained on the same medium or upon transfer of the regenerated shoot buds to medium containing 5.0 mg/l Kn and 1.0 mg/l IAA. Nearly a thousand callus regenerated plants were successfully transferred to the field following previously standardized hardening procedures.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichloro phenoxyaceticacid - 2iP 2-isopentenyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's medium (1962) - NAA -napthalene aceticacid - Z Zeatin     

In vitro micropropagation of the tropical fruit tree Syzygium cuminii L.

Multiple shoots were obtained from nodal and shoot tip segments of 10 to 15-day-old seedlings of Syzygium cuminii L. on Murashige & Skoog (MS) revised medium supplemented with 6-benzyladenine (BA) (0.23–8.90 M) singly or in combination with -naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). Excised shoots were placed for root induction on MS medium containing NAA and/or IBA and then transferred to MS basal medium to form complete plantlets. The regenerated plantlets have been acclimatized and successfully transferred into the soil.     

In vitro micropropagation of elite rosewood (Dalbergia latifolia Roxb.)

Induction of single and multiple shoots was obtained from nodal expiants of 60–80 year-old elite trees of rosewood on Murashige and Skoog's basal medium supplemented with 6-benzylaminopurine (1.0 mg 1^-1) and -Naphthalene acetic acid (0.05 mg 1^-1) or indole acetic acid (0.5 mg 1^-1). Multiplication of shoots was obtained on MS (reduced major elements) or Woody Plant Medium supplemented with 6-benzylaminopurine (1.0 mg 1^-1) and kinetin (0.5–1.0 mg 1^-1). Excised shoots were rooted on half-strength MS with IBA (2.0 mg 1^-1) to obtain complete plantlets. The regenerated plantlets have been acclimatized and successfully transferred to the soil.Abbreviations MS Murashige and Skoog's (1962) medium - B5 Gamborg (1968) medium - WPM Woody plant medium, Lloyd and McCown (1981) medium - NAA -naphthalene acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BAP 6-benzyl aminopurine - KIN kinetin - PVP polyvinyl pyrrolidone - CH casein hydrolysate - ADS adenine sulphate - L-Gl L-glutamine - L-Arg L-arginine - L-Asp L-asparagine - PG phloroglucinol     

Tissue culture and plant regeneration from immature embryo explants of Calotropis gigantea (Linn.) R. Br.

Callus cultures were established from immature embryos of Calotropis gigantea (Linn.) R. Br. on a modified basal medium of Murashige & Skoog supplemented with 1 mgl^-1 2,4-D. In addition to 0.1 mgl^-1 of NAA the optimal BAP concentration for promoting shoot bud formation and growth was 2 mgl^-1. Rooting was induced when shoots were transferred to auxin-supplemented Bonner's solution or half-strength MS basal salt solutions.Abbreviations NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-butyric acid - BAP 6-benzylaminopurine - Kin kinetin     

Clonal propagation of Picrorhiza kurroa royle ex benth. by shoot tip culture

A procedure has been developed for the clonal propagation of Picrorhiza kurroa Royle ex Benth. through shoot tip culture. Murashige and Skoog's medium (1962) supplemented with kinetin (3.0 to 5.0 mg/l) supported rapid proliferation of multiple shoots from the explants. Addition of indole-3-acetic acid (1.0 mg/l) to the kinetin containing medium showed marked improvement in the growth of regenerated shoots. However, presence of IAA in the medium did not alter the frequency of shoot multiplication. Rooting was readily achieved upon transferring shoots onto MS medium containing -naphthaleneacetic acid (1.0 mg/l). Plantlets were successfully transferred to soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid     

Clonal propagation of Cinnamomum zeylanicum Breyn. by tissue culture

Multiple shoot formation was induced directly from seeds of Cinnamomum zeylanicum Breyn. and also from seedling explants on Murashige and Skoog's medium containing different concentrations and combinations of auxins and cytokinins. Individual shoots were excised and induced to root on White's liquid medium. These plantlets were then transferred to pots in the green house and were eventually grown successfully under field conditions. Explants from the nodal region of these in vitro rooted plants were also subcultured to fresh medium. They produced a new crop of multiple shoots which could again be rooted by the same procedure.Abbreviations BAP 6-Benzylamino purine - 2,4-D 2,4-Dichlorophenoxy acetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid - KN Kinetin - NAA -Naphthalene acetic acid     

Tissue culture and plant propagation of Gomphrena officinalis — a Brazilian medicinal plant

Leaf and stem segments of Gomphrena officinalis originated from aseptically grown seedlings were used to initiate cultures. Callus production was obtained on gelled Murashige & Skoog medium supplemented with 6-benzylaminopurine alone (1.0, 5.0 or 10.0 mgl^-1) or combined with -naphthalene acetic acid (0.1, 0.5 and 1.0 mgl^-1) after 10 to 15 days of culture, and can be transferred to fresh medium every 30 days. The combinations of 5.0 or 10.0 mgl^-1 of 6-benzylaminopurine with 0.1 mgl^-1 of -naphthalene acetic acid were found to be the best for shoot regeneration. Adventitious shoot formation occurred after 50 to 60 days of culture in leaf and internode stem explants. Nodal segments developed actively growing lateral buds after 30 days of culture. Gelled Murashige & Skoog medium containing 10 mgl^-1 of indole-3-butyric acid was considered optimal for the rooting of shoots. Rooted plants transferred to potting soil could be successfully established.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog - NAA -naphthalene acetic acid     

Morphogenetic potential of foliar explants in Duboisia myoporoides R.Br. (Solanaceae)

The effects of serial combinations of either indole-3-acetic acid, indole-3-butyric acid or -naphthaleneacetic acid (0.5–10.0 mg/l) with either kinetin, 6-benzyl-amino-purine, zeatin or 6-methylaminopurine (0.5–5.0 mg/l) have been investigated to assess the morphogenetic potential of foliar explants of Duboisia myoporoides. Shoot buds developed either directly or via a callus interphase. Combinations involving indole-3-acetic acid with any of the cytokinins were more effective in inducing shoot bud formation compared to those containing indole-3-butyric acid or -napthalenacetic acid as an auxin. Among cytokinins, zeatin, kinetin and 6-benzylamino-purine were equally effective for shoot formation. However, optimum response with zeatin could be achieved at low concentrations (0.5–2.0 mg/l), while kinetin and 6-benzylamino-purine exhibited comparable efficacy at higher levels (3.0–5.0 mg/l). 6-Methylaminopurine proved least effective in all concentrations and combinations tested. Rooting of the differentiated shoots was readily achieved with -naphthaleneacetic acid alone (0.5 mg/l) after changing the physical form of the medium from gel to static liquid. Regenerated plantlets were transferred to pots and grown to maturity in the field with a high rate of survival (80–90%).Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MAP 6-methylaminopurine - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - PVP polyvinyl pyrrolidone     

Somatic embryogenesis and plant regeneration in a woody species: the European Spindle Tree (Euonymus europaeus L.)

Somatic embryogenesis and subsequent plant regeneration of Euonymus europaeus L (European Spindle Tree) were obtained from square pieces of mature zygotic embryos with an intervening callus phase. Callus and somatic embryos were induced using a Murashige and Skoog's semi-solid basal medium supplemented with several combinations of auxins and cytokinins. The greatest number of somatic embryos was obtained with a continuous exposure to 22.8 M indoleacetic acid and 0.046 M kinetin. The frequency of somatic embryogenesis from zygotic embryos depends on the cold conservation time of seeds. The embryos frequently germinated on the same medium. Further development of somatic embryos into plantlets was achieved on a medium devoid of growth regulators.Abbreviations MS Murashige and Skoog's medium - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - BAP 6-benzylaminopurine - RH relative humidity     

Effects of ascorbic acid on axillary shoot induction in Tylophora indica (Burm. f.) Merrill.

A procedure for rapid in vitro multiplication of Tylophora indica (Burm. f.) Merrill., an important indigenous medicinal plant, has been developed. Addition of ascorbic acid was essential to induce sprouting of axillary buds. Optimum multiplication was observed on MS medium containing 6-benzylamino purine (5.0 mg l^–1), -naphathalene-acetic acid (0.5 mg l^–1) and ascorbic acid (100 mg l^–1). Rooting of in vitro produced shoots was readily achieved with indole-3-acetic acid alone (1.0 mg l^–1) in MS. The plantlets thus obtained were successfully transferred to pots in large numbers which grew normally.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2ip 2-isopentenyladenine - Kn kinetin - MS Murashige & Skoog media - NAA -naphthalene acetic acid     

Clonal propagation of papaya in vitro

A procedure for the rapid tissue culture propagation of papaya is being developed. Tissue culture methods using apices of nursery and orchard trees of Carica papaya cv. Sunrise Solo were evaluated. The explants were established in a modified Murashige and Tucker (1969) basal medium with half-strength inorganic salts, 0.5mgl^-1 6-benzylaminopurine (BA) and 0.2mgl^-1 naphthaleneacetic acid (NAA). Established explants were transferred to a proliferation medium consisting of Murashige and Tucker (1969) basal medium, 0.5mgl^-1 BA and 0.1mgl^-1 NAA, which caused extensive multiplication of shoots. Rooting was induced at a higher frequency by subculturing plantlets onto media with indole-3-butyric acid (IBA) than with NAA.     

Organogenesis and tuberization in cultures of Dioscorea floribunda

Callus cultures were established from node and internode segments of Dioscorea floribunda Mart. & Gal. Both Murashige and Skoog's and modified White's medium supported callusing as well as organogenesis when supplemented with either 2,4-D or NAA in combination with BAP or Kn. On development of shoot primordia, calli were transferred to unsupplemented, half strength MS basal medium. This procedure led to the increase in formation of shoots. Several crops of shoots were obtained from single differentiating callus cultures by excising the shoots and subculturing the residual part. Seventy percent of plantlets survived rooting and transfer to soil.When they were maintained in half-strength MS basal medium and 0.5 mg1^-1 of NAA, 70% of plantlets formed aerial tubers at nodes. These tubers produced both roots and shoots and could be detached from the mother plant.     

In vitro micropropagation from nodal segments of Cleistanthus collinus

Cleistanthus collinus Benth. was micropropagated using nodal explants on MS medium supplemented with 2.2 M benzyladenine (BA). April to June was the best time for initiating shoot cultures. Shoot proliferation was enhanced when the BA concentration was lowered to 1.1 M. Rooting was achieved on half-strenth MS medium with 22.8 M indole-3-acetic acid for 7 days and continuous darkness for the first 72 h of the 7 days.Abbreviations MS Murashige & Skoog's medium - WPM Woody Plant Medium - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthalenacetic acid     

In vitro high frequency plant regeneration from hypocotyl and root segments of spinach by organogenesis

An efficient protocol for spinach (Spinacia oleracea L.) plant regeneration from hypocotyl and root segments was established. When the sub-apical hypocotyl and tip-free root segments were cultured on Murashige & Skoog (1962)-based medium containing high concentrations of indole-3-acetic acid (85.62 M) and gibberellic acid (100 M), more than 75% and 90% of the hypocotyl and root explants, respectively, formed shoots. After elongation, more than 92% of the shoots rooted on medium supplemented with 2.85–5.71 M of indole-3-acetic acid. More than 70% of rooted plantlets survived in soil and were fertile. Significant interactions between growth regulator combinations, explant types and environmental conditions on shoot initiation, development and rooting were discussed.Abbreviations BA benzyladenine - BM Murashige & Skoog basal medium - B5 Gamborg et al. medium (1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - 2ip isopentenyladenine - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige & Skoog medium (1962) - NAA naphthaleneacetic acid - HS hypocotyl segments - RSS root segments of seedlings - RSV foot segments of in vitro plantlets     

Development of shoots and roots in anther-derived callus of Azadirachta indica A. Juss.—a medicinal tree

Callus originated in microsporangial wall layers and connective tissues of anthers containing uninucleate microspores on Nitsch's or Murashige and Skoog's medium supplemented with growth regulators. A higher percentage of cultures (43) produced callus on Nitsch's medium containing 10 M indole-3-acetic acid + 1 M 6-benzyladenine. After 13–15 weeks, green nodular structures and prominent roots developed in 25% of the cultures on Murashige and Skoog's medium + 10 M -naphthaleneacetic acid + 1 M kinetin. Multiple shoots were induced in this anther-derived callus when subcultured on Murashige and Skoog's medium augmented with 4.44 M 6-benzyladenine + 0.53 M -naphthaleneacetic acid along with 18.75 M polyvinylpyrrolidone. The excised shoots formed roots after subculturing on Murashige and Skoog's medium + 4.90 M indole-3-butyric acid + 18.75 M polyvinylpyrrolidone, thus developing complete plantlets. Examination of callusing anthers also revealed two- to multi-celled pollen masses with intact exine.Abbreviations BA 6-benzyladenine - CW coconut water - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - HCl hydrochloric acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KMnO₄ potassium permanganate - MS Murashige & Skoog's medium - NAA -naphthaleneacetic acid - NB Nitsch's medium - PVP polyvinylpyrrolidone     

In vitro propagation of prairie gentian

Explants of shoot tips, internodal stem sections, and leaf segments of Lisianthus, Eustoma grandiflorum (Griseb.) Schinners, Dwarf Purple were cultured in vitro on modified Murashige and Skoog (MS) media. Explants of shoot tips and internodal stem sections developed into multiple shoots, whereas, leaf segments turned chlorotic on a medium supplemented with 3 mgl^-1 benzyladenine (BA) and 0.2 mgl^-1 naphthalene acetic acid (NAA). Shoot proliferation was obtained on shoot tips and leaf segments with 3 mgl^-1 BA, but internodal stem sections became necrotic and died on this medium. Rooting was induced in cultures with multiple shoots by subculturing explants on a half-strength MS medium supplemented with 2 mgl^-1 indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to soil.     

Plant regeneration via somatic embryogenesis in ginger

Embryogenic callus cultures of ginger were induced from young leaf segments taken from in vitro shoot cultures. Among the four auxins tested in Murashige & Skoog medium, dicamba at 2.7 M was most effective in inducing and maintaining embryogenic cultures. Efficient plant regeneration was achieved when embryogenic cultures were transferred to Murashige & Skoog medium containing 8.9 M benzyladenine. Histological studies revealed various stages of somatic embryogenesis characteristic of the monocot system. The in vitro-raised plants have been established in soil.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid     

Morphogenetic responses of cultured cells of cambial origin of a mature tree — Dalbergia sissoo Roxb

Regeneration of plantlets was achieved from cell suspension derived calli of cambial origin from mature elite trees of Dalbergia sissoo. Callus proliferation occurred on the cambial tissue pieces cultured on MS medium supplemented with 2, 4-dichlorophenoxyacetic acid (2.0 mg/1) and benzylaminopurine (0.1 mg/l). Suspension cultures were obtained by transferring and agitating callus lumps in liquid medium composed as above. Aggregates of about 30 cells were plated on semi solid medium, which developed into calli. Shoot bud differentiation was observed in the calli transferred to medium devoid of auxin but containing 0.5–2.0 mg/1 benzylaminopurine. The isolated microshoots were rooted on modified MS medium containing low organic salts and auxins.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IPA indole-3-propionic acid - KN kinetin - MS Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid