Somatic embryogenesis and in vitro flowering of 3 species of bamboo

Plant regeneration via somatic embryogenesis was achieved in callus cultures derived from nodal explants of in vitro grown seedlings and excised mature zygotic embryos of three bamboo species on Murashige and Skoog's (MS) basal medium supplemented with 0.5 mg/l kinetin (Kn), 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 10 mg/l adenine sulphate (Ads) and 3% (w/v) sucrose incubated in the light or in the dark. Somatic embryos germinated (95–98%) into normal plants and were transferred to soil with 95% success. In vitro flowering was induced on shoots developed from nodal explants taken from somatic embryo regenerated plants of Bambusa vulgaris, Dendrocalamus giganteus and Dendrocalamus strictus on half-strength MS basal medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA), 0.5 mg/l Ads, 0.5 mg/l gibberellic acid (GA₃) and 3% sucrose.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - Ads adenine sulphate - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - GA₃ gibberellic acid     

Induction of pollen callus in anther cultures of Feijoa sellowiana Berg. (Myrtaceae)

Summary Anthers of Feijoa sellowiana Berg. (feijoa) produced pollen callus when cultured in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid and benzyladenine or in nurse cultures. Somatic callus was also formed in large amounts from the connective and from the cut end of the filament. Anthers containing microspores at the stage immediately prior to the first pollen mitosis cultured in the presence of 3% sucrose, presented the highest frequencies of induction. Androgenetic divisions were initiated by the formation of two morphologically equal cells, the so-called B-pathway. Attempts to regenerate pollen plants were unsuccessful but leaf-like structures could be obtained in regeneration media containing combinations of gibberellic acid and benzyladenine.Abbreviations 2,4-D 2,4-diclorophenoxyacetic acid - BA benzyladenine - FDA fluorescein diacetate - GA₃ gibberellic acid - Kn kinetin - MS Murashige and Skoog (1962) medium     

A highly efficient in vitro regeneration methodology for mature Chinese tallow tree (Sapium sebiferum Roxb.)

A highly efficientin vitro regeneration methodology for mature chutese tallow tree (Sapium sebiferum Roxb.) has been developed. Shoot segments cultured on MS medium supplemented with 7.5 µM NAA produced light green callus. Optimum shoot differentiation resulted when callus was transferred to MS medium with 1 µM BA and 0.25 µM NAA. Shoot forming ability of callus was higher on MS medium compared to B5, half-MS or WPM. A continuous shoot harvest system at four-week intervals was established. Shoot yield continued for six months without loss of vigour. Regenerated shoots were rooted by culturing on half strength agar-gelled MS medium containing 1 µM IBA. Rooted plantlets were transferred to 1:1 soil vermiculite mixture and acclimatized with 67 % survival rate. Fully acclimatized plants were planted in the field, and performance is being evaluated.Abbreviations 2, 4- D 2, 4-dichlorophenoxyacetic acid - NAA 1 - napthaleneacetic acid - BA benzyladenine - Kn kinetin - 2-ip 6 - (, -dimethylallylamino)-purine - IBA indole-3-butyric acid - WPM woody plant medium (1980) - MS Murashige and Skoog (1962) medium - B5 Gamborg et al's medium (1968)     

Formation of plantlets through somatic embryogeny in the Himalayan blue poppy,Meconopsis simplicifolia (Papaveraceae)

Summary Exuberant and subculturable calli could be induced from only hypocotyl and leaf segments of ca 4-month-old seedlings of Meconopsis simplicifolia cultured on Murashige & Skoog's medium supplemented with 10^–6M kinetin + 10^–5M -naphthalene acetic acid. Suspension cultures were initiated from the calli in a similar medium but with 10^–5M 2,4-dichlorophenoxy acetic acid in place of -naphthalene acetic acid. In ca 80% of the suspension cultures somatic embryos differentiated freely (80–85%) as well as on the surface of small clumps of tissue (15–20%). Somatic embryos that developed beyond heart-shaped stage were transferred to agar-solidified Murashige & Skoog's medium free of growth substances. When maintained in 10 h light and 14 h dark the somatic embryos developed into plantlets bearing cauline leaves. From seed sowing to raising normal plantlets via callus required 28 weeks; on average 80 plantlets were obtained from one explant in three passages.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - Kn kinetin - MS Murashige & Skoog's medium (Murashige and Skoog 1962) - NAA -naphthalene acetic acid     

Somatic embryogenesis and subsequent plant regeneration from inflorescence callus of Bambusa beecheyana Munro var. beecheyana

Somatic embryos of bamboo, Bambusa beecheyana Munro var. beecheyana were developed in callus derived from young florets and adventive roots obtained from floret callus. The medium was a modified Murashige and Skoog medium (1962) supplemented with 3 mg/l 2,4-dichlorophenoxyacetic acid, 2 mg/l kinetin, a high content of sucrose (6%) and 0.7% agar. The embryoids germinated spontaneously to yield whole plantlets on this medium with or without the hormonal adjuvants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - MS Murashige and Skoog's (1962) medium     

Plant regeneration from mesophyll protoplasts of Williams' Bon Chretien (syn. Bartlett) pear (Pyrus communis L.)

Leaf protoplasts of axenic shoot cultures of Pyrus communis L. cv. Williams' Bon Chretien (syn. Bartlett) underwent cell wall regeneration and division to give multicellular colonies in a modified Murashige and Skoog medium which lacked ammonium ions, but supplemented with 1-naphthaleneacetic acid (NAA), 4-indole-3yl-acetic acid, 6-benzylaminopurine (BAP) and casein hydrolysate. Protoplast-derived colonies gave callus on Murashige and Skoog salts medium with NAA and BAP and exhibited shoot regeneration on half-strength Murashige and Skoog medium supplemented with 0.2 mg 1^–1 4-indole-3yl-butyric acid, 2.0 mg 1^–1 BAP, 0.2 mg 1^–1 gibberellic acid, 50 mg 1^–1 casein hydrolysate and 10 mg 1^–1 Ca-pantothenate. Following rooting, protoplast-derived plants of pear were transferred to the glasshouse where they completed acclimatization.Abbreviations BAP 6-benzylaminopurine - FPE final plating efficiency - GA₃ gibberellic acid - IAA 4-indole-3yl-acetic acid - IBA 4-indole-3yl-but yric acid - IPE initial plating efficiency - NAA 1-naphthaleneacetic acid - f.wt. fresh weight - MES 2-N-morpholinoethane sulfonic acid - MS Murashige and Skoog (1962) - %PE % plating efficiency - PVP-10 polyvinylpyrrolidone (Av. MW 10,000) - FDA fluorescein diacetate     

Rapid propagation of lemongrass (Cymbopogon flexuosus (Nees) Wats.) through somatic embryogenesis in vitro

Somatic embryos induced from callus cultures of lemongrass [Cymbopogon flexuosus (Nees) Wats.] on Murashige and Skoog medium supplemented with 5 mg/l of 2,4-D, 0.1 mg/l of NAA and 0.5 mg/l of Kn developed into plantlets when plated on a medium supplemented with 3 mg/l of BA, 1 mg/l of GA₃ and 0.1 mg/l of NAA. The regeneration potential of callus was retained for more than 2 years on the nutrient medium supplemented with comparatively lower levels of growth regulators (2,4-D at 2 mg/l, NAA at 0.1 mg/l and Kn at 0.25 mg/l). Approximately 30–35 plantlets were produced after two months of culture per 100 mg of callus inoculated. Regenerants were transplanted into soil and transferred to the field for assessment of various morphological and biochemical characteristics. The results of 1 year of field trials showed that plants derived from somatic embryoids were more uniform in all the characteristics examined when compared with the field performance of plants raised through slips by standard propagation procedures. Thus, a procedure has been developed for high frequency long term plant production of lemongrass through in vitro methods.Abbreviations 2,4-D 2,4 -dichlorophenoxyacetic acid - NAA -naphthalene acetic acid - Kn kinetin - BA benzyladenine - GA₃ gibberllic acid - MS Murashige and Skoog (1962) basal medium     

Plant regeneration by somatic embryogenesis from cultured immature embryos of oak (Querem robur L.) and linden (Tilia cordata Mill.)

Embryogenic cultures and somatic embryos were obtained from immature zygotic embryos of oak (Quercus robur L.) cultured on a modified MS medium and WPM containing BAP (1 mg·l^–1) and GA₃ (1 mg·l^–1) or BAP and IBA. Germination and conversion of oak somatic embryos into plantlets was achieved on WPM containing a reduced concentration of cytokinin. Linden (Tilia cordata Mill.) somatic embryos developed in embryogenic tissues initiated from immature zygotic embryos cultured on a modified MS medium supplemented with 2,4-D (0.3-2.0 mg·l^–1). Germination of linden somatic embryos and plantlet formation occurred on MS medium containing a low concentration of IBA. Oak and linden plantlets produced from somatic embryos were successfully established in soil. Somatic embryos and plantlets were also regenerated from embryogenic cultures of Quercus petraea and Tilia platyphyllos.Abbreviations BAP 6-benzyIaminopurine - GA₃ gibberellic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - WPM woody plant medium     

Micropropagation of a fruit tree, Morus australis Poir. syn. M. acidosa Griff.

High frequency bud break and multiple shoots were induced in nodal explants collected between November to February from a 5 year old tree of Morus australis Poir syn. M. acidosa Griff. on Murashige and Skoog's medium supplemented with 6-benzylaminopurine (1.0 mg/1). Incorporation of gibberellic acid (0.3 mg/l) along with BAP (1.0 mg/l) not only induced faster bud break from nodal explants as well as from apical shoot buds, but it also enhanced the frequency of bud break. Nodal explants were more responsive than apical shoot buds. The shoots formed in vitro were multiplied further as nodal segments, and an average multiplication rate of 6-fold per subculture was established within 4–5 months. The shoots were successfully rooted on half-strength MS containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/1. The plantlets were successfully hardened off and established in natural soil.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - KN kinetin - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA indole-3-propionic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalene acetic acid     

In vitro callus induction and plant regeneration from leaf explants of Ruta graveolens L.

A protocol for multiple shoot bud induction and plant regeneration from leaf segment-derived callus of Ruta graveolens has been developed. Maximum organogenic callus induction frequency (70.6 ± 2.33%) was observed on Murashige and Skoog (MS) medium supplemented with 10 µM 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Multiple shoot induction was achieved from the surface of the callus when transferred to shoot induction media (MS nutrients supplemented with 6-benzyladenine (BA), kinetin (Kn), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-naphthalene acetic acid (NAA) in various concentrations and combinations). The highest shoot multiplication (92.3%) was observed on MS medium with 7.5 µM BA and 1.0 µM NAA. Regenerated shoots were rooted in vitro on MS containing 0.5 µM IBA. Plantlets with well developed root and shoot systems were successfully acclimated (90%) and established in earthen pots containing garden soil; they exhibited normal morphology and growth characteristics.     

Plant regeneration from protoplasts of Panax ginseng (C.A. Meyer) through somatic embryogenesis

Summary Protoplasts of Panax ginseng were isolated from embryos obtained from the 4-year old embryogenic cell line KCTC PCL 49031 which was derived from a zygotic embryo. High protoplast yields of 22–25 × 10⁶ protoplast / g tissue were obtained following 5–6 h digestion with 2% Cellulysin, 1% Pectinase and 1% Macerasae in half strength Murashige and Skoog's medium containing 12% mannitol. A plating density of 1×10⁵ protoplasts /ml was found optimal for protoplast culture. An initial division frequency of 10% was obtained in an agarosegelled defined medium. Myo-inositol (6%) was found to be the most suitable osmoticum. Somatic embryos were formed from protoplast derived embryogenic callus, which regenerated into plantlets.Abbreviations NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn kinetin - BA benzyladenine - GA₃ gibberellic acid - MS Murashige and Skoog medium     

In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil)

A procedure is outlined for in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/l for O. americanum and 1.0 mg/l for O. sanctum. Incorporation of 0.5 mg/l gibberellic acid (GA₃) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoot buds collected between September through December were most responsive in culture. Shoots of O. americanum were rooted on half-strength MS supplemented with 1.0 mg/l indole-3-butyric acid (IBA), whereas O. sanctum rooted best on medium with 1.0 mg/l naphthaleneacetic acid (NAA). The plantlets were hardened off and successfully established in natural soil, where they grew and matured normally.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Plant Regeneration Through Somatic Embryogenesis in Leaf Derived Callus of Plumbago Rosea

Regeneration of Plumbago rosea L., a rare medicinal plant, via somatic embryogenesis in callus cultures derived from leaf explants was described. Optimum callus formation was achieved on semi-solid Murashige and Skoog (MS) medium supplemented with 0.25 mg dm^–3 kinetin and 2.0 mg dm^–3 1-naphthaleneacetic acid (NAA). Somatic embryogenesis was achieved upon transferring the 4-week-old callus to a medium containing 1.0 mg dm^–3 kinetic (Kn), 0.5 mg dm^–3 gibberellic acid (GA₃) and 0.1 mg dm^–3 NAA. Embryo maturation and germination was achieved on the half-strength MS basal salts supplemented with 0.01 – 0.25 mg dm^–3 Kn and 2 % (m/v) saccharose. An average of 50 – 60 plantlets were obtained from 150 mg of embryogenic callus within 4 week of subculture. Out of the 50 plantlets about 28 survived in the greenhouse.     

In vitro propagation of herbaceous peony (paeonia lactiflora pall.) by a longitudinal shoot-split method

A procedure for the clonal propagation ofPaeonia lactiflora Pall. cvs. Takinoyosooi and Sarah Bernhardt through shoot tip culture is described. Half strength Murashige and Shoog (1962) medium supplemented with 0.5 mg/l 6-benzylaminopurine plus 1 mg/l gibberellic acid promoted formation and growth of axillary buds. Continuous shoot multiplication was achieved by vertically splitting the shoot axis and subsequent division of elongated axillary shoots every 36 days. High frequency (57–100%) of rooting was obtained on paper-bridge liquid medium supplemented with 1 mg/l indole-3-butyric acid. Half of the rooted plantlets were established on porous soil. Thus, 700 and 300 plants of cv. Takinoyosooi and Sarah Bernhardt could be theoretically obtained from a single bud in one year.Abbreviations BAP 6-benzylaminopurine - GA gibberellic acid - NAA a-naphthaleneacetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) basal medium     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.     

Stimulation of in vitro shoot proliferation from nodal explants of cassava by thidiazuron, benzyladenine and gibberellic acid

Multiple shoots were produced from nodal explants of cassava (Manihot esculenta Crantz) by a two-step procedure: a 6- to 8-day exposure to 0.11–0.22 µM thidiazuron (TDZ) in liquid Murashige and Skoog (MS) medium followed by culture on agar-solidified MS medium supplemented with 2.2 µM 6-benzyladenine (BA) and 1.6 M gibberellic acid (GA₃). TDZ caused the nodal explants to expand and this expansion (growth) continued during culture with BA and GA₃. From this expanded explant, clusters of buds and fasciated stems developed continuously and these gave rise to shoots. The shoot proliferation process was open-ended, yielding an average of 31.5 shoots per nodal explant after 10 weeks of culture with genotype CG 1–56. A positive response was also obtained from seven other genotypes evaluated with this protocol.Abbreviations BA 6-benzyladenine - BM basal medium - DPU 1,3-diphenylurea - GA₃ gibberellie acid - 2iP isopentenyladenine - MSM multiple shoot medium - NAA 1-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - Z zeatin     

Culture of and fertile plant regeneration from regenerable embryogenic suspension cell-derived protoplasts of wheat (Triticum aestivum L.)

Summary Regenerable embryogenic cell suspensions initiated from immature embryo-derived friable, fast growing, embryogenic calli of GK Ságvári winter wheat (Triticum aestivum L.) served as sources of protoplasts, which were cultured in different liquid or agarose-solidified media. Protocallus formation was best on KM_8p (Kao and Michayluk 1975) and GM (Li and Murai 1990) media, and protocallus growth on MS (Murashige and Skoog 1962) callus growing medium. Green shoot/plant regeneration occurred on MS regenerating medium, and rooting on MS or N6M (Mórocz et al. 1990) hormone-free media. Protocalli maintained their morphogenic capacity over 4 months, and with multiple subcultures on half-strength MS regenerating medium, the total number of regenerants could be increased. Approximately 1000 shoots/plants were regenerated and over 500 plants were transplanted in the greenhouse. The majority of them had an abnormal chromosome number and low viability, however, one plant grew to maturity and set seed.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - GA₃ gibberellic acid - GM General medium - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog medium - NAA 1-naphthaleneacetic acid - RECS regenerable embryogenic cell suspension     

Somatic embryogenesis and plant regeneration from protoplasts of asparagus (Asparagus officinalis L.)

Protoplasts were isolated from embryogenic calli of Asparagus officinalis L. cv. Mary Washington and cultured in 1/2 MS medium with 1 mg/l NAA, 0.5 mg/l zeatin, 1 g/l L-glutamine, 0.6 M glucose and 0.1% Gellan Gum. Protoplasts started to divide after 3–4 d of culture and formed visible colonies after 30 d of culture. The percentage of colony formation (plating efficiency) was 7.2%. The colonies were then transferred onto Gellan Gum-solidified MS medium containing 1 mg/l 2,4-D and 3% sucrose for further growth. Somatic embryos were induced from all colonies of 0.5–1.0 mm size after transferring to 1/2 MS medium lacking growth regulators. After treating these somatic embryos (1–3 mm) in distilled water for a week, 30–40% of them germinated normally and grew into plantlets 20–30 d after transplanting on 1/2 MS medium containing 1 mg/l IBA, 1 mg/l GA₃ and 1% sucrose. These protoplast-derived plants were diploid with 20 chromosomes.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962)     

Morphogenic potential of mechanically isolated single cells from Digitalis obscura L. callus

Calli from hypocotyl and root explants of Digitalis obscura L. showed regeneration of adventitious shoots, roots and embryos when transferred to Murashige & Skoog medium supplemented with cytokinins alone or in combination with auxins. Optimum shoot-bud formation was achieved in the presence of IAA and BA, while roots mainly appeared either in absence of growth regulators or with IAA and Kn. Embryo formation took place only in those combinations that included Kn. Embryo development was influenced by the type of auxin, and precocious germination occurred in media with NAA. Mechanically isolated cells from hypocotyl- and root-derived calli were plated in MS medium supplemented with several IAA and BA combinations. Single cells were able to proliferate forming callus within 20–30 days in culture. In order to induce organogenesis, calli were transferred to various regeneration media. Shoot-bud differentiation efficiency depended on both callus origin and medium initially used for cell culture, best results being obtained in calli grown from hypocotyl-derived cells cultured in the presence of casein hydrolysate. A further subculture to medium containing coconut milk and lower concentrations of NH₄NO₃ and sucrose promoted shoot development. Rooting was readily achieved upon transferring shoots onto half-strength MS medium. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - BM basal medium - CH casein hydrolysate - CM coconut milk - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - Kn kinetin - MS Murashige & Skoog - NAA naphthaleneacetic acid     

Micropropagation of Hemidesmus indicus (L.) R. Br. through axillary bud culture

A procedure for rapid in vitro propagation of the aromatic and medicinal plant Hemidesmus indicus (L.) R.Br. (Family Asclepiadaceae) from nodal explants is described. The highest shoot multiplication rate of 8.2 ± 0.4 shoots/explant with a 95% frequency was achieved in S weeks culture period on Murashige and Skoog medium supplemented with 1.15 M kinetin and 0.054 M -naphthaleneacetic acid. Excised shoots were rooted on the same basal medium supplemented with 1.15 M kinetin and 7.35 M indole-3-butyric acid. Shoots derived from subcultures exhibited better rooting response than those from primary cultures. After a hardening phase of 2 weeks, there was a 70% transplantation success in the field.Abbreviations MS Murashige and Skoog (1962) medium - BA N⁶ benzyladenine; KN kinetin - NAA a-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid