Resistance to freezing in liquid nitrogen of carnation (Dianthus caryophyllus L. var Eolo) apical and axillary shoot tips excised from different aged in vitro plantlets

The ability of shoot tips from carnation (Dianthus caryophyllus L., var. Eolo) cultured in vitro to develop resistance to freezing in liquid nitrogen depends on the physiological state of the cell material and the pretreatment conditions. Regrowth rates close to 100% have been obtained with apical shoot tips isolated from 2 month-old stems, precultured on medium supplemented with sucrose (0.75M) and treated with dimethylsulfoxide (5% or more). Resistance of axillary shoot tips decreased progressively as a funtion of their distance from the apical shoot tip. During the development of the stem from axillary buds (obtained by cutting), progressive increases in the regrowth rate of frozen apices were noted, from 30% before cutting (axillary buds) to 98% after 3 weeks of culture.Abbreviations DMSO dimethylsulfoxide - LN liquid nitrogen     

In vitro propagation through axillary bud multiplication in different mulberry genotypes

Axillary buds from 5 genotypes of mulberry belonging to 4 species were cultured on modified MS basal medium. A total of 30 media combinations were tried for all the genotypes. The response of axillary buds and the requirement for growth regulators varied with genotype. In Morus indica BAP (0.25–0.5 mg/l), and in M. alba and M. rotondifolia GA₃ (0.5–1.0 mg/l)were found to induce sprouting. Two genotypes of M. bombycis, namely Schimanochi and Mizusawa, developed healthy shoots on the incorporation of 2,4-D (0.5–1.0 mg/l) and BAP (0.5–2.0 mg/l), respectively. IBA (0.5 mg/l), along with cytokinin/auxin/gibberellin, had no effect on bud growth but helped root induction. Shoots developed from the axillary buds were further multiplied as nodal explants. MS basal medium supplemented with 0.5 mg/l IBA and LS vitamins was found best to produce healthy plantlets in all the genotypes. An average 89% survival was observed on transferring the plantlets to soil.Abbreviations MS Murashige and Skoog (1962) - LS Linsmaier and Skoog (1965) - IBA 3-indole-butyric acid - GA₃ Gibberellic acid - BAP 6-Benzylaminopurine - Kn Kinetin - 2,4-D 2,4-Dichlorophenoxyacetic acid     

In vitro plant regeneration of leguminous trees (Albizia spp)

Hypocotyl explants of three leguminous forest tree species, Albizia amara, A. lucida and A. richardiana, have differentiated shoot buds on B₅ basal medium. Maximum number of shoots per explant developed on basal medium augmented with 2,4-D (0.1 M) in A. amara (2) and BA (10 M) for both A. lucida (2) and A. richardiana (1.6). Higher concentrations of auxins in the medium, in general, enhanced rooting and callusing but cytokinins promoted the growth of green calli. BA enchanced the differentiation of shoots in the three species. The in vitro grown shoots of A. amara and A. richardiana, after subculturing on B₅+1 M IAA developed roots (up to 30–40%). These plants have been successfully transferred to the field.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzylaminopurine - BM Gamborg's B₅ medium with 0.9% agar+3% sucrose - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - NAA -naphthaleneacetic acid     

Regeneration of plants from the culture of leaves and axillary buds in mulberry (Morus indica L.)

Stem segments, axillary buds and leaves excised from established shoot cultures of Morus indica were soaked in MS liquid medium containing benzyladenine (0.5, 1, 2 mg/1) and were cultured subsequently on semi solid medium of the same composition. Numerous shoot buds differentiated from leaf and axillary buds but stem segments were unresponsive. The shoot buds on isolation and culture developed into plantlets. Callus tissues which developed at the base of the leaf explant upon subculture also differentiated numerous shoot buds.Abbreviations BA benzyl adenine - CM coconut milk - 2, 4-D 2, 4 dichlorophenoxy acetic acid - Kn kinetin - MS Murashige and Skoog - Z zeatin     

Cryopreservation of encapsulated shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures

Shoot primordia induced inArmoracia rusticana Gaertn. Mey. et Scherb. (horseradish) hairy root cultures were successfully cryopreserved by two cryogenic procedures. Encapsulated shoot primordia were precultured on solidified Murashige-Skoog medium supplemented with 0.5M sucrose for 1 day and then dehydrated with a highly concentrated vitrification solution (PVS2) for 4 h at 0°C prior to a plunge into liquid nitrogen. The survival rate of encapsulated vitrified primordia amounted to 69%. In a revised encapsulation-dehydration technique, the encapsulated shoot primordia were precultured with a mixture of 0.5M sucrose and 1M or 1.5M glycerol for 1 day to induce dehydration tolerance and then subjected to air-drying prior to a plunge into liquid nitrogen. The survival rate of encapsulated dried primordia was more than 90%, and the revived primordia produced shoots within 2 weeks after plating. A long-term preservation of shoot primordia was also achieved by the technique. Thus, this revised encapsulation-dehydration technique appears promising as a routine method for the cryopreservation of shoot primordia of hairy roots.Abbreviations PVS2 Vitrification solution - LN liquid nitrogen - BA 6-benzyladenine - NAA -naphthalene-acetic acid - MS Murashige and Skoog (1962) medium     

Embryogenesis and plant regeneration from anther culture of bamboo (Sinocalamus latiflora (Munro) McClure)

Embryogenic callus was initiated from bamboo (Sinocalumus satiflora (Munro) McClure) anthers cultured on N₆ medium supplemented with 1 mg/l 2,4-D, 1 mg/l BA, 2 g/l charcoal, 0.8% agar (Sigma) and 9% sucrose. Anthers with microspores at miduninucleate to early-binucleate stages showed better rate of response for callus induction. Prolonged culture of these embryogenic calli on the original medium or subculture to an auxin-free medium resulted in embryoid formation and their subsequent germination to form rooted plantlets. Chromosome counts from root-tip cells of anther-derived plant indicated that they were haploid (N=36).Abbreviations N₆ Chu et al. (1975) - MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

Cryopreservation of non-encapsulated embryogenic tissue of sweet potato (Ipomoea batatas)

Embryogenic tissue of the sweet potato (Ipomoea batatas (L) LAM) genotype TIB 10 was established from in vitro axillary shoot tips on Murashige and Skoog (1962) medium supplemented with 5 M 2,4-dichlorophenoxyacetic acid. Embryogenic aggregates of fresh mass 9.0–12 mg were subjected to a rapid freezing protocol in liquid nitrogen following sucrose preculture and varying degrees of dehydration. Up to 50% of embryogenic explants survived rapid freezing after preculture on 0.4 or 0.7M sucrose only. Dehydration with silica gel to moisture contents in the range 18–41% improved the survival after cryopreservation of embryogenic tissue. Tissue dehydrated for intermediate periods exhibited poor survival. Following freezing, embryogenic tissue appeared to develop normally, retaining its competence to produce mature embryos and plantlets.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Rapid clonal propagation of Pinellia ternata by tissue culture

Adventitious buds or protocorm-like bodies were regenerated directly from excised explants without intervening callus. Differences in the ability of regeneration were observed among different plant organs with bulbils showing the highest regenerative ability followed by leaf blade and petiole. Ability of vegetative propagation of bulbil could be maintained by alternate solid-and liquid-medium culture. Theoretically, 1.7×10²⁷ plantlets could be produced from a single bulbil by this technique within one year based on the production and rapid growth of protocorm-like bodies and adventitious buds. Concentration of MS salts, NAA and sucrose influenced not only root formation from the differentiated adventitious buds, but also root number and length. For root formation, the best combination was one-half strength MS salts with 3–5% sucrose and 1 mg/l NAA. The high survival rate of 96% was recorded when plantlets were transplanted into a mixture of vermiculite:loam soil:peat moss (1:2:1). Plants from in vitro culture were morphological similar to field-grown plants. The acute toxicity of crude extracts from protocorm-like bodies was about one-fourth that of extracts from tubers of field-grown plants when tested with white mice. Tissue culture has potential for clonal propagation of Pinellia ternata plants for commercial use.Abbreviations MS Murashige and Skoog (1962) - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BA 6-benzylaminopurine     

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 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     

Anther culture of some perennial triticeae

Three field grown Agropyron spp. (crested wheatgrasses) and two Thinopyrum spp. (intermediate and tall wheatgrasses) were evaluated for anther culture response. Hormonally modified potato extract and 85D12 media induced pollen embryogenesis. Modified Murashige and Skoog media were tested for their effects on callus proliferation and plantlet regeneration. Callus induction frequency and plantlet production were highest (25.0% and 45.8%, respectively) for Thinopyrum ponticum (2N=70) (tall wheatgrass). One-hundred and nine albino plantlets were produced from T. ponticum Jose both by direct regeneration on 85D12 medium and through a callus phase from potato extract media. This is the first report of plantlet production from anther culture of a Triticeae perennial forage grass. Further experimentation with environmental and cultural conditions may result in the production of green plantlets.Abbreviations MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-ip 2-isopentenyladenosine - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid Cooperative investigations of the USDA-Agricultural Experiment Station and the Utah Agricultural Experiment Station, Logan, UT 84322. Approved as Journal Paper No. 3596     

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     

Multiple shoot production from seedling explants of slash pine (Pinus elliottii,Engelm.)

Hypocotylary explants obtained from 30- to 40-day-old slash pine (Pinus elliottii, Engelm.) seedlings treated with 6-benzylaminopurine produced multiple buds that eventually elongated into axillary shoots. The explants were pulse treated (45-s dip) with 6-benzylaminopurine (22.2, 111, 222 M) plus a control and cultured on three different basal media containing activated charcoal (0.5% w/v). Hormonal concentration and basal medium were compared for the number and size of axillary shoots induced after 12 and 29 days. The greatest number of axillary shoots was produced by explants that were pulse treated with 111 M 6-benzylaminopurine and cultured on Gresshoff and Doy medium. The axillary shoots were fewer in number per explant than shoots previously reported resulting from hormonally induced advantitious buds of slash pine, but the axillary shoots developed more rapidly.Abbreviations BA 6-benzylaminopurine - DMSO dimethyl sulfoxide - PAR photosynthetically active radiation     

Clonal propagation of Leptospermum spp. by tissue culture

Propagation by axillary and multiple axillary bud development was achieved in three native Leptospermum spp. when axillary buds derived from nodal tissues ex mature plants were placed in benzylaminopurine media (0.04–1.0 M) containing macro- and micro-nutrients, sucrose (0.06 M) and a vitamin/amino acid supplement. Reduction of agar concentration from 0.8 to 0.2% greatly stimulated axillary bud development and growth in L. flavescens and L. brachyandrum. Rooting of axillary shoots was stimulated by 2,4-dichlorophenoxyacetic acid and p-chlorophenoxy acetic acid in L. flavescens at concentrations of 5 and 1 M respectively. In L. petersonii ssp. root initiation and development was favoured by -naphthoxyacetic acid (1 M) and in L. brachyandrum indole butyric acid and -naphthalene acetic acid (1 M) were almost equally effective.     

Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - PVS2 vitrification solution - LN liquid nitrogen - BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - SE standard error - ABA abscisic acid     

In vitro selection of endosulfan-tolerant strains of Brassica compestris (cv. Brown Sarson)

Endosulfan tolerant lines of mustard (Brassica campestris cv. Brown Sarson) have been developed through tissue culture methods. Cotyledonary expiants excised from eight day old in vitro grown seedlings were used for inducing callus. Fast growing friable callus was then transferred to MS medium containing (0.1–2.0 ugl^–1) endosulfan for selection. Five alternating exposures with and without endosulfan containing medium yielded an endosulfan tolerant cell line (ETL). The plants regenerated from ETL were found to tolerate three fold higher concentrations of endosulfan. Callus induced from randomly selected endosulfan tolerant regenerated plants were also tolerant to 3.0 ugl endosulfan, thereby, suggesting that tolerance has been acquired at the gene level.Biochemical investigation revealed higher levels of total free sugar, free amino acids, protein and activity of peroxidase in the tolerant cell line.Abbreviations MS Murashige and Skoog medium (1962) - NSM non-selective medium - SM selective medium - BAP 6-Benzylaminopurine - NAA -naphthaleneacetic acid - Z zeatin     

Somatic embryogenesis and organogenesis of Agave victoriae-reginae: Considerations for its conservation

Agave victoriae-reginae somatic embryos were produced through a callus phase from seedling stem segments cultured on MS medium. The optimal treatment was MS medium with 2.26 M 2,4-D. Multiple shoot regeneration was induced from axillary buds from stem segments cultured on MS medium with 2.2–4.4 M BA. Effect of MS and modified MS medium with 50% macronutrient concentration, both containing 2.2 M BA and sucrose at the following concentrations, 20, 30, 45 and 60 g l^–1, resulted in inconsistent multiple shoot formation. Shoots and somatic embryos formed by this indirect pathway could have a multicellular origin, which might lead to genetic variation. The direct development of pre-existent buds occurred on MS basal medium and increased in the presence of BA; this might be a pathway for the rescue of genotypes of endangered species. Embryos and shoots developed and grew roots on MS medium. Complete plantlets were obtained on MS basal medium. A total of 92% per cent of the plantlets survived and grew when transferred to the greenhouse. Agave micropropagation could supply the commercial plant demand, diminishing the gathering of seeds and plants of this endangered species from the wild.     

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

Seeds of trifoliate orange (Poncirus trifoliata (L.) Raf.) are sensitive to desiccation, and could not withstand reduction in moisture level below 20%, whereas the excised embryonic axes could be easily desiccated to moisture levels as low as 14% without much loss in viability. Axes could be successfully cryopreserved in liquid nitrogen (–196°C) for eight months. The viable embryonic axes exhibited good growth on modified Murashige and Skoog medium supplemented wiith 1-Naphthalene acetic acid (NAA) and 6-Benzylaminopurine (BAP). Growth of cryopreserved axes was promoted in the presence of charcoal in the medium allowing for plant recovery.Abbreviations NAA Napthaleneacetic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) - LN Liquid nitrogen     

Plant regeneration from Carica protoplasts

Summary Rapidly proliferating and highly regenerable suspension cultures of somatic embryos of Carica papaya x C. cauliflora were used for protoplast isolation. On average, protoplast yield was 1.5×10⁶/g fresh weight of somatic embryos. Protoplasts were first cultured in liquid KM8P-S medium for 2 weeks and then plated in the same medium solidified with 1% agarose. About 1.4% of the protoplasts developed directly into somatic embryos. Protoplast-derived somatic embryos proliferated rapidly through direct embryogenesis on modified MS medium supplemented with 1 mg/1 ABA, and developed into plantlets upon transfer to MS medium devoid of plant growth regulators. The plantlets were successfully transplanted to soil.Abbreviations MS Murashige and Skoog medium (1962) - KM8P Kao and Michayluk medium (1975) - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - CPW Frearson et al. medium (1973)