Clonal propagation byin vitro culture of Corchorus (jute)

Callus was produced on cotyledon, shoot tip, hypocotyl and root explants of twoCorchorus species on several media. Cytokinin was necessary for callus production on cotyledon explants. BothC.olitorius genotypes produced most callus on media with zeatin and either NAA or IAA, and theC.capsularis genotype produced most callus on media with IAA and either zeatin or BA. High frequencies of regenerated shoots were obtained from shoot tip explants of both species, from the apical meristem and from callus. Media with 2.0 mg 1⁻¹ BA were superior for both species, and media with zeatin were equally good forC.capsularis only. More regeneration was obtained for all genotypes after subculture of callus on media with 2.0 mg 1⁻¹ zeatin. Cotyledon callus produced less regeneration, also with differences between genotypes; explants of both genotypes ofC.olitorius produced regeneration on a medium with NAA and zeatin, and theC.capsularis genotype produced regeneration on a medium with IAA and BA. Limited regeneration from root explant callus was obtained forC.capsularis only on medium with BA and IAA. Regeneration was not obtained from hypocotyl callus. Further regeneration of shoots of both species was obtained from secondary callus after subculture, and from nodal segments of regenerated shoots and of seedling shoots cultured on basic MS medium without growth hormones. Roots were produced on about 80% of all shoots after transference to medium with 0.2 mg 1⁻¹ IBA, and rooted plantlets survived and flowered normally after transference to compost.     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

Transformation of Solanum integrifolium poir via Agrobacterium tumefaciens: Plant regeneration and progeny analysis

Summary The wild species Solanum integrifolium represents a source of pest and disease resistance genes for breeding strategies of the cultivated species Solanum melongena. Somatic hybridization via protoplast fusion between the two species may provide a valuable tool for transferring polygenic traits into the cultivated species. The availability of S.integrifolium cells carrying dominant selectable markers would facilitate the heterokaryon rescue. An appropriate methodology for in vitro culture and plant regeneration from leaf explants of S.integrifolium is reported. Efficient leaf-disk transformation via co-cultivation with Agrobacterium tumefaciens led to the regeneration of transformed plants carrying the reporter genes GUS and NPT-II. Transformed individuals were obtained through selection on kanamycin-containing medium. Stable genetic transformation was assessed by histochemical and enzymatic assays for GUS and NPT-II activity, by the ability of leaf disks to initiate callus on Km-containing medium, Southern blot analyses of the regenerated plants, and genetic analysis of their progenies. Selfed-seed progeny of individual transformed plants segregated seedlings capable to root and grow in selective condition, while untransformed progeny did not. Genetic analyses of progeny behaviour showed that the reporter gene NPT-II segregated as single as well as two independent Mendelian factors. In two cases an excess of kanamycin-sensitive seedlings was obtained, not fitting into any genetic hypothesis.Abbreviations MS Murashige and Skoog (1962) medium - NOS nopaline synthase - NPT-II neomycin phosphotransferase - GUS beta-glucuronidase - LB Luria and Bertani medium - KIN 6-furfurylaminopurine - BAP 6-benzylaminopurine - 2iP N⁶-(2-isopentyl)adenine - ZEA zeatin - TDZ Thidiazuron     

Adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (<Emphasis Type="Italic">Crataegus pinnatifida</Emphasis> Bge. var. <Emphasis Type="Italic">major</Emphasis> N.E.Br.)

Hawthorn (Crataegus spp.) is an important plant with a long history as an ornamental and a source of medicine. A protocol is outlined for adventitious bud regeneration from leaf and cotyledon explants of Chinese hawthorn (C. pinnatifida Bge. var. major N.E.Br.). Adventitious buds were induced on both the leaves of sprouting winter buds and the leaves of in vitro plants, but the percentage of bud regeneration from leaves of in vitro plants was very low—less than 6%. On N6 medium supplemented with 31.08 μM BA and 9.67 μM NAA, the percentages of bud regeneration from leaves of sprouting winter buds of cultivars “Liaohong” and “Qiujinxing” were 31.4% and 17.6%, respectively. The regeneration abilities of three kinds of cotyledon explants, immature cotyledon, mature cotyledon, and cotyledon leaf, were compared. The percentage of bud regeneration from cotyledon leaves was higher. On MS media supplemented with 4.44 μM BA and 4.54–9.08 μM TDZ, the percentages of bud regeneration from cotyledon leaves of cultivars “Qiujinxing” and “Xiajinxing” were 27.7 ± 7.8% and 20.1 ± 4.7%, respectively, and the numbers of buds per explant were 5.9 ± 1.6 and 3.2 ± 0.7, respectively. On B5 medium supplemented with 2.22 μM BA, 2.32 μM Kn, and 0.57 μM IAA, adventitious buds grew quickly and 80–100% of buds developed into shoots. The shoots rooted successfully with the two-step rooting method. Ninety days after transplantation, more than 80% plants were survived. This system of adventitious bud regeneration from leaf and cotyledon explants could be useful for the genetic transformation and polyploidization of Chinese hawthorn.     

Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid     

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     

In vitro plant regeneration from leaf and cotyledon explants of Cucumis melo L.

Five different genotypes from in vitro as well as greenhouse grown melon plants were shown to be highly responsive for in vitro shoot formation from leaf explants when placed on basic MS medium supplemented with 1 mg/l 6-benzylaminopurine. In addition, a very suitable regeneration system was obtained when cotyledon pieces of mature seeds were incubated on the same culture medium. In this case, the first shoots already appeared after 10 days of incubation, and hundreds of shoots were formed on the cut surface 3 to 4 weeks later. Explants from mature cotyledons derived from seedlings did not lead to any shoot formation.Abbreviations MS Murashige and Skoog - IAA 3-indoleacetic acid - BA 6-benzylaminopurine     

Efficient and genotype-independent Agrobacterium--mediated tomato transformation

An efficient method to transform five cultivars of tomato (Lycopersicon esculentum), Micro-Tom, Red Cherry, Rubion, Piedmont, and E6203 is reported. A comparison was made of leaf, cotyledon, and hypocotyl explants on 7 different regeneration media without Agrobacterium tumefaciens cocultivation and on 11 different media with cocultivation. Although all cultivars and explants formed callus and regenerated on the initial 7 media, cocultivation with A. tumefaciens significantly reduced the callus induction and regeneration. From these experiments, a transformation methodology using either hypocotyls or cotyledons cultured for one day on BA 1 mgL-1, NAA 0.1 mgL-1 and 3 days cocultivation with the Agrobacterium on this same medium followed by a transfer to a medium with zeatin 2 mgL-1 and IAA 0.1 mgL-1 for 4-6 weeks resulted in a greater than 20% transformation frequency for all five cultivars tested. In this transformation method, no feeder layers of tobacco, petunia or tomato suspension cultures were used, and the subculture media was minimal. Stable integration and transmission of the transgene in T1 generation plants were confirmed by Southern blot analysis. This procedure represents a simple, efficient and general means of transforming tomato.     

Transformation of eggplant (Solanum melongena L.) using a binary Agrobacterium tumefaciens vector

Summary Kanamycin resistant plants of Solarium melongena L. (eggplant) cv. Picentia were obtained following the cocultivation of leaf explants with Agrobacterium tumefaciens. A disarmed binary vector system containing the neomycin phosphotransferase (NPTII) gene as the selectable marker and chloramphenicol acetyltransferase (CAT) as a reporter gene was utilized. In vitro grown plants were used as sources of explants to produce transgenic plants on selective medium containing 100 mg/l kanamycin. The transformation and expression of the foreign genes was confirmed by DNA hybridizations, leaf disc assays, and by measuring NPTII and CAT enzyme activities. This technique is simple, rapid, efficient, and transgenic eggplants of this commercial cultivar have been transferred to soil where they have flowered and set seed.Abbreviations CAT chloramphenicol acetyltransferase - MS Murashige and Skoog - NPTII neomycin phosphotransferase - NOS nopaline synthase - ZEA zeatin     

In vitro regeneration of Nicotiana africana plants from explants of different type and mesophyll protoplasts

Methods of in vitro cultivation of an African endemic species Nicotiana africana (Solanaceae) possessing some valuable traits have been elaborated. Influence of different concentrations of auxin (alpha-naphtaleneacetic acid) and cytokinins (6-benzylaminopurine, zeatin, thidiazuron) on morphogenesis and plant regeneration has been analysed using leaves and internodes as explants. The optimum method of regeneration of N. africana shoots in leaf explants is cultivation in the presence of 0.1 mg/l NAA and 1 mg/l BA; in internode explants--cultivation on the medium containing 0.5 mg/l NAA and 1 mg/l zeatin. The use of thidiazuron was the most effective at the concentration of 0.4 mg/l with subsequent transfer of explants to hormone-free medium. Method of isolation and culture of mesophyll protoplasts enabling to regenerate N. africana plants during 3-4 months has been proposed.     

In vitro regeneration of sesame (Sesamum indicum L.) from seedling cotyledon and hypocotyl explants

The goal of this study was to develop an efficient regeneration protocol to be used for genetic transformation of sesame. Published regeneration methods using benzyladenine (BA) and 1-naphthalene acetic acid (NAA) were unsuccessful for the cultivars used herein. Experiments were carried out using cotyledon and hypocotyl explants from the cultivar Mtwara-2. Later the optimised culture conditions were used to investigate the regeneration response of different genotypes. There was significant interaction between hormone treatments and macronutrients for shoot and root regeneration. Results also showed that shoot regeneration was significantly influenced by explant type. Shoots were only obtained from cotyledons whereas both cotyledons and hypocotyls could produce roots. Modified Murashige and Skoog (MS) medium with N6 macronutrients resulted in twice the shoot regeneration frequency obtained with ½MS macronutrients in the presence of thidiazuron (TDZ). The shoot regeneration frequency was significantly reduced when BA was used in place of TDZ. On shoot regeneration medium containing BA and NAA, only roots were formed. Replacing NAA with indole-3-acetic acid (IAA) greatly improved the regeneration of shoots. The optimum growth regulator combination for shoot regeneration was 20 μM TDZ together with 2.5 μM IAA, which gave a frequency of 63% and 4.4 shoots per regenerating explant for the best cultivar Ex-El. Genotypic differences were significant both for the number of explants regenerating shoots and the number of shoots produced per regenerating explant.     

Plant regeneration from protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of six cultivars of Glycine max L. and cultured in the KP8 liquid medium supplemented with 0.2 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L ZT. The protoplasts started to divide after 3–5 days of culture. Sustained divisions resulted in mass production of cell colonies and small calli in 6 weeks. The calli further grew to 2–3 mm on the gelritesolidified K8 medium and were transferred onto the MSB medium with 1 mg/L 2,4-D and 0.25 mg/L BA, to obtain compact and nodular calli. Shoot formation was initiated on MSB medium with 0.15 mg/L NAA, and BA, KT and ZT, 0.5 mg/L of each, with or without 500 mg/L CH. It was followed by plant regeneration. So far, 87 plants have been regenerated from 4 cultivars, and normal seeds were obtained from them after transplanting into pots.Abbreviation IAA indol-3-acetic acid - NAA naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - KT kinetin - BA 6-benzyladenine - ZT zeatin - CH casein hydrolysate     

Influence of the antibiotic timentin on plant regeneration of tomato (Lycopersicon esculentum Mill.) cultivars

 Cotyledon explants of tomato (Lycopersicon esculentum Mill. cvs 'Santa Clara', 'Firme' mutant, 'IPA-5' and 'IPA-6') were excised from 8- to 10-day-old in vitro-grown seedlings. Four different shoot induction media supplemented with timentin (300 mg l^–1) were screened. When cotyledon explants were cultured on MS-based medium with 1.0 mg l^–1 zeatin plus 0.1 mg l^–1 IAA and supplemented with timentin, higher regeneration frequencies and a greater number of elongated shoots were obtained. It was observed that timentin caused an increase in the morphogenesis of in vitro cotyledon explants of tomato cultivars. In two of three cultivars tested, rooting of shoots was positively influenced, both in the presence and absence of timentin in the rooting medium, among shoots regenerated from explants derived from timentin-supplemented medium. The results confirm those of a previous investigation on the beneficial effects of this class of antibiotics on tomato regeneration and, consequently, its reliability for use in the transformation of this species. Received: 1 December 1998 / Revision received: 19 May 1999 · Accepted: 3 May 1999     

High efficiency plant regeneration from cotyledons of watermelon (Citrullus vulgaris Schrad)

Cotyledons of various ages from seedlings of eight watermelon (Citrullus vulgaris) cultivars were cultured on MS medium supplemented with different combinations of phytohormones. High frequency shoot regeneration (60.0–92.0%) was induced from 5-day-old cotyledons of cultivars cultured on MS medium containing 5.0 mg/l 6-benzylaminopurine (BA) and 0.5 mg/l indole-3-acetic acid (IAA). Multiple shoot buds elongated on MS medium containing 0.2 mg/l kinetin (KT) and 5–10 shoots per expiant could be recovered depending on the cultivars. Elongated shoots rooted on MS medium with 0.1 mg/l -naphthalene acetic acid (NAA). Zeatin riboside (ZT) had a similar efficiency as BA in shoot induction, and it was significantly more functional than 2-isopentenyladenine (2iP) or kinetin (KT). Cotyledons from 5-day-old seedlings were the most responsive to shoot induction.Abbreviation BA 6-benzylaminopurine - GA₃ gibberellic acid - IAA indole-3-acetic acid - 2iP 2-isopentenyladenine - KT kinetin - MS Murashige and Skoog (1962) - NAA -naphthalene acetic acid - ZT zeatin riboside     

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     

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.     

Embryogenesis and plant regeneration of Medicago spp. in tissue culture

Ten cultivars and breeding lines from two species of alfalfa (Medicago media and M. sativa) were screened for their ability to produce embryos and plantlets from the root and hypocotyl under three different tissue culture protocols. The three protocols differed in basal salt composition, vitamins, hormones and cytokinin additions. That protocol having a high 2–4,D low cytokinin induction step gave the highest percentage of embryogenic calli in some cultivars and lines. M. media cultivars and breeding lines had a high percentage of embryoid formation. M. sativa cultivars gave no embryoid formation. Two M. media breeding lines (Br1 and Le1), which were intermediate in the percentage of embryogenic calli formed from explants, had the highest number of regenerated plants established in soil. The creeping rooted M. media cultivar Heinrichs produced the highest percentage of embryogenic calli from explants but most of these embryoids were abnormal and failed to grow in soil or vermiculite. Accordingly, successful regeneration is directly related to the quality and quantity of the embryoids produced. Respectively: Biotechnology Department, Alberta Research Council, Agriculture Canada, Beaverlodge, Alberta, and University of Alberta, Edmonton, Alberta, Canada     

Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens

The leaf disc transformation/regeneration system was modified for tomato (L. esculentum). Both leaf explants and cotyledon/hypocotyl sections can be used to regenerate transformed plants. We have obtained over 300 transgenic plants from eight tomato cultivars. We have evidence for both single and multi-copy insertions of the T-DNA, and have demonstrated inheritance of the T-DNA insert in the expected Mendelian ratios. Several heterologous promoters function in tomato. A reduced efficiency of transformation was observed with binary T-DNA vectors as compared to co-integrate T-DNA vectors. The ease of the leaf disc method makes tomato a premier experimental organism for plant biotechnology.Abbreviations BA benzyl adenine - IAA indole acetic acid - LB Luria Broth     

Alkaloid production in Catharanthus roseus cell cultures

Callus derived from hypocotyls of periwinkle, Catharanthus roseus, responded to culture on nutrient media supplementedwith IAA, BA, and zeatin with shoot formation at low frequencies. However, shoot regenerating callus could be very successfully propagated and subcultured. Alkaloid profiles of callus derived from the original explants (hypocotyls) as well as callus derived from regenerated shoots were almost identical. Subcultures of old callus (initiated in 1978) failed completely to grow shoots. In programs for long-term preservation of alkaloid producing cell lines by regeneration and storage of shoots, selection for ability to form shoots would have to precede selection for alkaloid production.Abbreviations IAA indolyl-3-acetic acid - IIAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzyladenine NRCC No. 20087     

Plant regeneration from mesophyll protoplasts in commercial potato cultivars (Primura,Kennebec, Spunta,Desirée)

In view of an evaluation of the relative efficiency of different in vitro systems for mutant induction and isolation in potato, a procedure of plant regeneration from protoplasts of some potato (Solanum tuberosum L.) cultivars was developed. Four cultivars (Primura, Kennebec, Spunta, Desirée) were used for isolation, culture and regeneration of leaf mesophyll protoplasts.These lines were chosen because of their economic importance in Italy and in the case of cv. Desirée for the presence of markers useful in the morphological characterization of regenerants.Abbreviations MS Murashige and Skoog's medium - BAP 6-benzylaminopurine - 2,4 D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - GA ₃ gibberellic acid - ZT zeatin