Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana

Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.Abbreviations MS medium Murashige and Skoog's medium (Murashige and Skoog 1962) - B5 medium Gamborg B5 medium (Gamborg et al. 1968) - BA 6-benzylaminopurine - TDZ N-phenyl-N'-1,2,3-thiadiazol-5-yl urea - 4PU-30 N-(2-chloro-4-pyridyl)-N'-phenylurea - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid     

Conditions of transformation and regeneration of `Induka' and `Elista' strawberry plants

Efficiency of plants' transformation depends on many factors. The genotype, applied techniques and conditions of plant's modification and modified plant regeneration are the most important among them. In our studies regeneration and transformation conditions for two strawberry cultivars were determined and compared. Plants were transformed by Agrobacterium tumefaciens LBA₄₄₀₄ strain containing plasmid pBIN19 with nptII and gus-reporter genes. Experiment was carried out on more than 1300 leaf explants from each cultivar. Generally, `Induka' plants characterized with higher regeneration potential than `Elista'. The highest number of regenerated shoots was obtained on MS medium with 0.4 mg l ^–1 IBA and 1.8 mg l^–1 BA (3.5 and 1.8 shoots/explant for `Induka' and `Elista', respectively). After plant transformation number of regenerated, transgenic shoots was higher for `Elista' (on the average: 8.3 shoots/100 explants). The number of transgenic `Induka' shoots, obtained at the same conditions, was twice lower (4.2). Simultaneously `Induka' plants needed higher kanamycin concentration for transgenic explants selection than `Elista' (25 mg l^–1). Preliminary incubation of A. tumefaciens in LB or MS medium with acetosyringone and IAA resulted in increasing transgenic shoots number (per 100 explants: `Induka' 4.5, `Elista' 8.0–9.5 shoots). After using untreated bacteria for plants' transformation, number of transgenic plants varied (dependently on cultivar) from 3.8 to 7.0/100 explants. Applying LB or MS as basic medium as well as adding tobacco plant extract to these media did not significantly influence transformation efficiency.     

Synergistic effect of ethylene inhibitors and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey) in vitro

Summary The role of ethylene and putrescine on shoot regeneration from hypocotyl explants of Chinese radish (Raphanus sativus L. var. longipinnatus Bailey cv. Red Coat) was investigated. Explants were recalcitrant in culture, but exogenous application of ethylene inhibitor [20–30 M aminoethoxyvinylglycine (AVG) or AgNO₃] enhanced shoot regeneration of explants grown on medium supplemented with 2 mg/l N⁶-benzyladenine and 1 mg/l 1-naphthaleneacetic acid. The best regeneration occurred in the medium containing AgNO₃ in combination with AVG. Culture medium solidified with agarose in the presence of AgNO₃ but not AVG was also beneficial to shoot regeneration. Exogenous putrescine, 2-chloroethylphosphonic acid and 1-aminocyclopropane-1-carboxylate had no effect on shoot regeneration. However, regeneration was greatly promoted by 10–25 mM putrescine in combination with 30 M AgNO₃ or AVG. Explants with high regenerability grown in the presence of AgNO₃ or in combination with putrescine emanated high levels of ethylene throughout the 21-d culture period. By contrast, AVG or putrescine alone resulted in a decrease in ethylene production. For rooting of shoot cuttings, IAA and IBA at 1–5 mg/l were more effective than NAA.Abbreviations ACC 1-aminocyclopropane-1-carboxylate - AVG aminoethoxyvinylglycine - BA N⁶-benzyladenine - CEPA 2-chloroethylphosphonic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAs polyamines - SAM S-adenosyl-L-methionine     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of the heavy metal tolerant and hyperaccumulator <Emphasis Type="Italic">Arabidopsis halleri</Emphasis> (Brassicaceae)

Plants were regenerated from root explants of Arabidopsis halleri (L.) O’Kane and Al-Shehbaz via a three-step procedure callus induction, induction of somatic embryos and shoot development. Callus was induced from root segments, leaflets and petiole segments after incubation for 2 weeks in Murashige and Skoog medium (MS) supplemented with 0.5 mg/l⁻¹ (2.26 μM) 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.05 mg/l⁻¹ (0.23 μM) kinetin. Only calli developed from root segments continued to grow when transferred to a regeneration medium containing 2.0 mg/l⁻¹ (9.8 μM) 6-γ-γ-(dimethylallylamino)-purine (2ip) and 0.05 mg/l⁻¹ (2.68 μM) α-naphthalenacetic acid (NAA) and eventually 40 of them developed embryogenic structures. On the same medium 38 of these calli regenerated shoots. Rooting was achieved for 50 of the shoots subcultured in MS medium without hormones. The regeneration ability of callus derived from root cuttings, observed in this study, makes this technique useful for genetic transformation experiments and in vitro culture studies.     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

Multiple Shoot Induction from Cotyledonary Node Explants of Terminalia chebula

A protocol for multiple shoot induction from cotyledonary node explants of Terminalia chebula Retz. has been developed. Germination frequency of embryos (up to 100 %) was obtained on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^–3 gibberellic acid (GA₃). Maximum number of shoots (6.4 shoots per cotyledonary node) was obtained on half-strength MS + 0.3 mg dm^–3 GA₃+ 1.0 mg dm^–3 indole-3-butyric acid (IBA) + 10.0 mg dm^–3 benzylaminopurine (BAP) after 4 weeks of culture. When the cotyledonary nodes along with the axillary shoot buds were allowed to grow in the same medium upto 19.2 shoots were obtained after 8 – 9 weeks. Best rooting (100 %, 5.5 roots per shoot) was observed when shoots were excised and transferred to half-strength MS medium containing 1.0 mg dm^–3 IBA + 1 % mannitol and 1.5 % sucrose. Survival of rooted plants in vivo was low (35 – 40 %) when they were directly transferred to soil in glasshouse. However, transfer to soil with MS nutrients and 1.0 mg dm^–3 IBA in culture room for a minimum duration of 2 weeks increased the survival percentage of plants to 100 %.     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

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     

An efficient in vitro plant regeneration system for the medicinal plant Teucrium stocksianum Boiss.

An efficient and rapid plant regeneration system via direct organogenesis was established for Teucrium stocksianum Boiss. (Lamiaceae), an endangered and valuable medicinal plant. Hypocotyl explants excised from seedlings germinated in vitro were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of kinetin and indoleacetic acid (IAA) to induce shoot formation. Differentiation of multiple shoots was initiated within 3 weeks of culture. Optimal regeneration was achieved on medium containing 3 mg/l kinetin and 0.5 mg/l IAA. This particular medium composition significantly improved the production of multiple shoots directly from hypocotyl explants compared to other combinations of plant growth regulators. Root induction was achieved on half-strength MS medium containing indole-3-butyric acid. Rooted plantlets were successfully acclimatized, with a survival rate of 75–80%. The protocol developed in this study could be used for long-term in vitro conservation and mass propagation of this species.     

Transgenic Brassica campestris Plants Expressing the gfp Gene

A protocol has been worked out for efficient regeneration and genetic transformation of summer rape (Brassica campestris L. var. oleifera). Cotyledons of the 5-day-old seedlings were transformed with Agrobacterium tumifaciens strain AGL cells comprising a binary vector with a selectable neomycin phosphotransferase II gene and a reporter gene encoding green fluorescent protein (GFP). Explants were cultured on a regeneration MS medium supplemented with ABA, and transformants were selected on the same medium (minus ABA and plus 70 mM AgNO₃ and 15 mg/l kanamycin). The frequency of shoot regeneration on explant petioles was 30–40%. Transgenic plants were identified by GFP fluorescence and by polymerase chain reaction and Western blotting analysis. The transformation efficiency was as high as 75% of the total number of regenerated shoots.     

Micropropagation of <Emphasis Type="Italic">Swertia chirata</Emphasis> Buch.-Hams. ex Wall.: a critically endangered medicinal herb

An efficient in vitro plant regeneration protocol for Swertia chirata Buch.-Ham. ex Wall (Gentianaceae), a critically endangered Himalayan medicinal herb, was developed using shoot tip explants derived from in vitro grown seedlings. Media with 2% sucrose and various types of hormones markedly influenced in vitro propagation of S. chirata. An in vitro shootlet production system using Murashige and Skoog (MS) medium with various hormones such as BAP, KN and TDZ was established. BAP at 1.0 mg/l and KN, 0.1 mg/l induced highest number of multiple shoots (42.16 ± 1.05) per explant. Micro-proliferated shoots were transferred to elongation medium amended with GA₃ (0.1 mg/l) and hormone free basal medium, after which they were transferred to rooting medium. The highest frequency of rooting (22.48 ± 1.08) was obtained in half-strength MS medium supplemented with NAA, 0.1 mg/l after testing with different auxins at various concentrations within 4 weeks of transfer to the rooting medium. Hardening was successfully attained under controlled conditions inside the plant tissue culture room. This method could effectively be applied for the conservation and clonal propagation to meet the pharmaceutical demands.     

Plant regeneration from tissue cultures of Persian buttercup (Ranunculus asiaticus L.)

Different plant explants of Persian buttercup (Ranunculus asiaticus L.) were screened for callus induction and adventitious shoot regeneration on different media to establish totipotent cultures. Murashige & Skoog (MS) medium was used, supplemented with different concentrations of the following growth regulators: kinetin, benzyladenine (BA), naphthaleneacetic acid (NAA) and indoleacetic acid (IAA). Callus was induced and adventitious buds regenerated only from cotyledonary explants after 4–5 weeks. Subculture of the regenerated buds on the same basal medium in presence of gibberellic acid (GA₃) and BA produced well-organized shoots. Rooting was obtained by transferring shoots to growth regulator-free MS medium. A high rate of shoot multiplication has been achieved on medium with high concentration of kinetin and long-day photoperiod. Finally the plants were successfully transferred to soil and grown in a greenhouse.     

AgNO3 boosted high-frequency shoot regeneration in Vigna mungo (L.) Hepper

In order to further increase shoot regeneration frequency of Vigna mungo (L.) Hepper., the effects of AgNO₃ on this process was investigated in this study. The shoot tip and cotyledonary node explants were cultured on MS salts B5 Vitamins medium containing BA+TDZ+Ads+AgNO₃ for multiple shoot induction. AgNO₃ influenced the shoot bud formation and their subsequent proliferation. The best medium composition for multiple shoot induction was BA, TDZ combination with Ads and AgNO₃ in MSB5 medium. Maximum 39 shoots in cotyledonary node and 22 shoots in shoot tip were obtained per explants after 4 – 6 wk. of culture. Elongation and rooting were performed in GA₃ (0.6mg/l) and IBA (0.4mg/L) containing media respectively. The in vitro raised plantlets were acclimatized in green house and successfully transplanted to the field with a survival rate of 78%.     

High frequency plant regeneration from cotyledon and hypocotyl explants of ornamental kale

A high frequency shoot regeneration system for ornamental kale [Brassica oleracea L. var. acephala (D.C.) Alef.] was firstly established from seedling cotyledon and hypocotyl explants. The ability of cotyledon and hypocotyl to produce adventitious shoots varied depending upon genotype, seedling age and culture medium. The maximum shoot regeneration frequency was obtained when the explants from cv. Nagoya 4-d-old seedlings were cultured on Murashige and Skoog (MS) medium supplemented with 3 mg dm⁻³ 6-benzylaminopurine (BA) and 0.1 mg dm⁻³ naphthaleneacetic acid (NAA). The frequency of shoot regeneration was 65.0 % for cotyledons, 76.1 % for hypocotyls; and the number of shoots per explant was 4.3 for cotyledons, 8.2 for hypocotyls. Hypocotyl explants were found to be more responsive for regeneration when compared with cotyledons. Among the 4 cultivars tested, Nagoya showed the best shoot regeneration response. The addition of 3.0 mg dm⁻³ AgNO₃ was beneficial to shoot regeneration. Roots were formed on the base of the shoots when cultured on half-strength MS medium.     

Clonal propagation of Acacia catechu Willd. by shoot tip culture

A method is described for in vitromicropropagation through shoot apices of Acaciacatechu Willd., a semi-arid tree valued for Katha (atanin-like substance obtained from red heart wood of10–20 year old trees) and timber. Explants wereexcised from 15-days-old in vitro grownseedlings raised from superior seed stocks. Shoot budinduction from shoot apex explants was observed onMurashige and Skoog's (MS) [12] medium containingvarious growth regulators. A maximum of 12 shoots wasobtained on MS medium supplemented with 1.5 mg/l 6-benzylaminopurine (BAP) and 1.5 mg/l kinetin.Well-developed shoots (3–4 cm long) were rooted on strength MS medium with 3.0 mg/l indole-3-acetic acid (IAA) and sucrose 1.5%. In vitro regenerated plantlets of A. catechu were transferred to field conditions.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Transgenic plants of rutabaga (Brassica napobrassica) tolerant to pest insects

Cotyledons cut from axenic seedlings were immersed inAgrobacterium tumefaciens suspension which was treated with acetosyringone and nopaline at low pH overnight. The infected cotyledon explants were cultured on MSB medium (MS salts + B₅ Vitamins) containing 6-BA 3mg/1 for 2–3 days, and transferred onto selective medium (MSB with kanamycin 50–100 mg/l). Kanamycin-resistant shoots were selected. More than 60 regenerated plants were obtained. About 60% of the plants showed high NPT II activity. Southern blot hybridization showed that some of the plants gave a positive signal with the insecticidal crystal protein gene (cry IA gene) probe, and exhibited tolerant to insects such asPieris rapae (cabbage caterpillar) in leaf feeding experiments. Kanamycin-resistance and insect-resistance were maintained in the progeny.Abbreviations 6-BA 6-benzylaminopurine - IBA indole-3-butyric acid - CryIA gene bacillus thuringiensis insecticidal crystal protein genecryIA - NPT II neomycin phosphotransferase II     

High frequency of plant regeneration from hypocotyl explants of Brassica carinata A. Br.

An efficient tissue culture system for high frequency of plant regeneration from hypocotyl explants of Brassica carinata was developed via manipulation of culture medium and selection of explants. Explants grown on medium containing combinations of 2 mg l^-1 BA and 0.01 mg l^-1 NAA or 4 mg l^-1 kinetin and 0.01 mg l^-1 2,4-D regenerated shoots at 100% frequency. High frequency shoot regeneration occurred only from explants originating from 6 to 7-day-old but not younger or older seedlings. Explants showed higher regeneration capacity at the distal end than the proximal end, and the upper segment was more regenerative than the lower segment of hypocotyl. Regenerants were rooted on half-strength growth regulator-free medium, acclimatized and developed into normal, fertile plants.Abbreviations BA benzyladenine - 2-4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog     

High frequency adventitious shoot regeneration from immature cotyledons of pea (Pisum sativum L.)

A procedure has been developed which allows high frequency adventitious shoot regeneration from immature cotyledons of pea. Prolific shoot regeneration occurred following an initial callus growth on a Murashige and Skoog (MS) medium containing 0.5 mg/l 6-benzylaminopurine (BAP) and 4 mg/l -naphthaleneacetic acid (NAA). Cotyledon expiants proximal to the embryonic axis had the highest regeneration potential, however, the presence of an embryonic axis inhibited adventitious shoot regeneration. Addition of silver nitrate (AgNO₃) to the medium did not promote the number of regenerated shoots but resulted in shoots with well developed tendrils and large stipules which had a reduced rooting capacity. Regenerated shoots rooted readily (80–90%) in half strength MS medium containing 1 mg/l indole-butyric acid (IBA) and further established well in compost.     

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.