High-frequency plant regeneration from cotyledon callus of <Emphasis Type="Italic">Acacia sinuata</Emphasis> (Lour.) Merr

Summary A new reliable protocol for the induction of adventitious shoots and plant regenertion from cotyledon-derived callus of Acacia sinuata has been developed. Calluses were induced from cotyledon explants on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar or 0.15% phytagel, 8.1 μM α-naphthaleneacetic acid, and 2.2 μM 6-benzylaminopurine (BA). High-frequency regeneration of adventitious buds from callus was achieved when cultured on half-strength MS medium supplemented with 10% coconut water, 13.3 μM BA, and 2.5 μM zeatin. Histological studies revealed that the regenerated shoots originated from the callus. Among the various carbohydrates tested, sucrose at 87.6 mM was optimum for shoot-bud induction. Addition of 1.7 μM gibberellic acid along with 4.4 μM favored shoot elongation. In vitro-raised shoots produced prominent roots when transferred to half-strength MS medium supplemented with 7.4 μM indole-3-butyric acid. Rooted plants, thus developed, were hardened and successfully established in soil (45%). This protocol yielded an average of 40 plantlets per cotyledon explant over a period of 3 mo.     

Rapid plant regeneration from various explants of Jatropha integerrima

A simple, rapid and reproducible protocol for direct shoot regeneration from different explants of Jatropha integerrima was developed. Prolific adventitious shoot bud initiation was obtained using a combination of 2.2 or 4.4 M benzyladenine and 4.9 M indole-3-butyric acid (IBA). Reduction of IBA concentration (2.5 M) promoted further development of shoots. Regenerated shoots rooted readily on Murashige and Skoog (MS) medium lacking growth regulators. Plantlets were acclimatized and successfully transferred to pots.     

Organogenesis and somatic embryogenesis in callus cultures ofRosa hybrida andRosa chinensis minima

Pre-incubation of somatic tissues of the cut rose Carefree Beauty and miniature roses Red Sunblaze and Baby Katie in 10, 100, or 200 M 2,4-D induced the development of highly rhizogenic callus. Upon transfer of rhizogenic callus to a regeneration medium containing 23 M TDZ and 3 M GA₃, a low frequency of shoot organogenesis (3.3%) and somatic embryogenesis (6.6%) was observed. Incubation of leaf and stem internodes in 11, 27, 54, 81, or 108 M NAA followed by transfer of explants to the regeneration medium resulted in up to a 25% increase in shoot organogenesis from callus-derived internodal explants of Red Sunblaze, but no somatic embryogenesis was observed. The influence of glucose versus sucrose in the regeneration medium on leaf explants of Carefree Beauty and Baby Katie pre-incubated in 100 M 2,4-D revealed a difference in genotypic response to shoot organogenesis and somatic embryogenesis. For Carefree Beauty, a concentration of 111 mM glucose induced higher frequencies of both organogenic (33%) and embryogenic calluses (25%) than either 59 mM or 117 mM sucrose. For Baby Katie, no significant difference was found for number of organogenic calluses induced on 59 mM sucrose and 111 mM glucose.Abbreviations ABA Abscisic acid - BA Benzyladenine - 2,4-D 2,4-Dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA Indoleacetic acid - MS Murashige & Skoog (1962) - NAA -Naphthaleneacetic acid - TDZ Thidiazuron     

矮牵牛花药培养及植株再生研究

采用花粉发育双核期的矮牵牛花药,研究不同浓度植物生长调节剂配比及不同浓度蔗糖和麦芽糖对花药诱导率的影响。结果表明,采用6-BA和IBA组合诱导效果较好;NH+6-BA 1.5mg/L+IBA 1.5mg/L花药诱导率较高;麦芽糖诱导效果明显比蔗糖好。     

High frequency plant regeneration from excised leaves ofPaulownia fortunei

Summary Rapid regeneration of multiple shoots ofPaulownia fortunei was obtained from the petiolar ends of leaf explants from in vitro grown shoots. The optimal shoot-inducing treatment was Murashige-Skoog medium supplemented with 4μM naphthaleneacetic acid and 20μM N⁶-benzyladenine. Shoot buds were visible in more than 80% of the explants, mainly from the petiolar cut ends, by 7 days in culture. Shoot growth was promoted by transferring explants to fresh medium once every 2 wk. As many as 43 shoots per explant were obtained in 13 wk. Regenerated shoots could be easily rooted and successfully transplanted to a peat-based potting mixture.     

Efficient plant regeneration of native spearmint (Mentha spicata L.)

Summary Protocols and media constituents for efficient in vitro plant regeneration of Native Spearmint (Mentha spicata L. cultivar ‘Native Spearmint’) have been defined. Adventitious shoots were initiated either directly from morphogenetically competent cells of explants or primary callus. Leaf explants from at least 2-mo.-old in vitro-maintained shoots exhibited the greatest morphogenetic capacity. Explants derived from basal portions of leaves at the bottom of the shoot were most responsive, with up to a 100% regeneration frequency and greater than nine shoots per explant. Highest frequency of meristemoids and morphogenetic callus were initiated from explants cultured onto a basal medium containing Murashige and Skoog (MS) salts, supplemented with 4 mg thidiazuron (TDZ) per L and 25% (vol/vol) coconut water (CW) for 10 to 14 d in darkness. Bud and shoot development required removal of both TDZ and CW from the medium. Shoot propagules were transferred to basal medium supplemented with 0.01 mg α-naphthaleneacetic acid (NAA) per L and grown under low light for about 2 wk to facilitate shoot elongation. Individual shoots about 1 cm tall were dissected and retransferred onto the same medium. Root initiation began within 4 to 6 d and a functional root system developed within 2 to 3 wk. These plantlets were transferred to soil and acclimated successfully for growth and development in a greenhouse. This is the first report of an efficient regeneration system for Native Spearmint based on adventitious organogenesis.     

紫色大花矮牵牛组织培养与植株再生

矮牵牛叶片外植体在MS+6-BA 1.0mg/L+NAA 0.1mg/L培养基上培养3周后产生致密的浅绿色愈伤组织;转入芽分化培养基MS+6-BA 0.5mg/L+4-PU 0.5mg/L+NAA 0.1mg/L 1周后,从愈伤组织表面不断分化产生幼芽;待幼芽长至3cm时转接至生根培养基1/2MS+NAA 1.0 mg/L+GA₃0.5mg/L中生根,长成完整植株。     

The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes

In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K⁺ uptake by roots. When F₁ hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.In the summer of 1984, immature embryos from 218 additional inbred lines and germplasm stocks were plated and examined for their ability to produce regenerable callus cultures on media containing altered micronutrient concentrations, 3,6-dichloro-o-anisic acid (dicamba), glucose, and elevated levels of vitamin-free casamino acids and thiamine. Of these genotypes 199 (91%) produced callus that was regenerable in appearance. In the 1984 study, plant regeneration was noted in many commercially important inbreds, including B73, Mo17, B84, A632, A634, Ms71, W117, H99³H95 and Cm105. Thus tissue-culture techniques are now available to obtain callus cultures capable of plant regeneration from immature embryos of most maize genotypes.Abbreviations trade names 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid     

Organogenesis plant regeneration from leaf explants of Exacum Styer Group

Exacum Styer Group plantlets were regenerated through direct organogenesis from leaf explants. Four genotypes were evaluated on MS media supplemented with combinations of BA (0, 0.44, 2.22, 4.44, or 8.88 μM) and NAA (0, 0.05, 0.54, or 2.69 μM) for direct shoot organogenesis without an intervening callus phase. Regression analyses were used to analyze and interpret the data. There were significant genotype, media, and genotype × media interactions for several variables. Genotypes 01-09-01 and 01-37-61 had the highest number of shoots per explant across media (10.2 and 6.6, respectively) while the 4.44 μM BA plus 0.54 μM NAA treatment induced the greatest number of shoots among the genotypes evaluated.     

Adventitious shoot regeneration from immature embryos of sorghum

Eleven genotypes of sorghum were examined for their response in tissue culture, and the tissue culture system was optimized. The cultures were initiated from immature embryos taken approximately two weeks after flowering. The response of immature embryos varied with the genotype. `C. Kafir' and `PE932 025' showed the highest frequency of callus induction and regenerable callus formation under appropriate culture conditions. Regeneration occurred at high frequencies when cytokinins (kinetin or 6-benzyladenine) had been added in the callus induction medium, followed by regeneration medium devoid of growth regulators. The addition of proline and polyvinylpyrrolidone also enhanced shoot formation, but the addition of cytokinins to regeneration media did not improve shoot formation. On the revised culture medium, plants were regenerated from up to 100% of sorghum immature embryos.     

Somatic embryogenesis and plant regeneration from immature zygotic embryos of papaya (Carica papaya L.)

Summary Immature zygotic embryos from open-pollinated and selfed Carica papaya L. fruits, 90 to 114 days post-anthesis, produced 2 to 20 somatic embryos on apical domes, cotyledonary nodes, and radicle meristems after culture for three weeks on half-strength Murashige and Skoog (MS) medium supplemented with 0.1 to 25 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 400 mg l^–1 glutamine, and 6% sucrose. After six weeks of culture, about 40 to 50% of the zygotic embryos had become embryogenic, and each embryogenic embryo yielded hundreds of somatic embryos within five months of culture on media supplemented with 2,4-D. Somatic embryos matured on half-strength MS medium, germinated on MS medium containing 5 mg l^–1 kinetin, and grew large enough for greenhouse culture on MS medium. Shoots were rooted in vermiculite and grown in the greenhouse.Journal Series no. 3449 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Callus formation and plant regeneration from immature and mature embryos of rye (Secale cereale L.)

Summary An efficient protocol was developed to regenerate entire plants from immature embryos of elite genotypes of rye as a prerequisite to plant transformation. Three winter genotypes and one spring genotype were tested using both immature and mature embryos as explants. Four types of callus initiation media and five kinds of regeneration media were tested in all possible combinations. Immature embryos gave much higher levels of plant regeneration than mature embryos, but mature embryos could be induced to regenerate plants for all genotypes and media tested, although at low levels. A minimum stage of embryo development must be reached before embryos can be cultured successfully. Genotypic effects were less pronounced than those reported for inbred cereal species such as wheat and barley, but there was an effect of genotype on percentage of callus formation. There was a significant interaction between genotype and initiation media. Composition of the initiation media affected both the percentage of callus formation from embryos and subsequent frequencies of plant regeneration. Composition of the regeneration media had no effect on level of plant regeneration. Immature embryos of all genotypes tested could be induced to produce 90–100% callus on appropriate initiation media and all regenerated shoots from approximately one-half to three-quarters of the calluses produced.     

Somatic embryogenesis and plant regeneration from immature cotyledons of watermelon

Cotyledon expiants from immature embryos of five watermelon [Citrullus lanatus (Thunb.)Matsum. & Nakai] genotypes were incubated in the dark for three weeks on a modified MS medium containing B5 vitamins, 2,4-D (10, 20 or 40M), 0.5 M of either BA or TDZ, and 7 g·1^-1 TC agar. Somatic embryos, some with well developed cotyledons, were observed on cotyledon expiants three to four weeks after transfer to MS medium without PGRs and 16h photoperiod. The best PGR combination for somatic embryogenesis was 10 M 2,4-D and 0.5 M TDZ Somatic embryogenesis was greatest (30%) when cotyledon expiants were established from 18-day-old immature embryos. Somatic embryos were germinated on MS medium without PGRs. Plants were transferred to Magenta boxes containing ProMix for three weeks before being transplanted to the field where they formed fertile male and female flowers that produced normal fruit.Abbreviations PGR plant growth regulator - BA benzyladenine - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyaceticacid - NAA -naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Adventitious shoot production from immature embryos of white clover

Cotyledonary-stage embryos of Haifa white clover, collected 13 days after cross-pollination, were induced to form adventitious shoots primarily from the hypocotyl region. The culture medium used for the production of adventitious shoots contained 5 M thidiazuron and 0.5 M -naphthaleneacetic acid. Numerous shoot meristems were produced within the first week, discrete shoots developed by week three, small plantlets by week eight, and whole plants in soil by week ten. 95–100% of all embryos, regardless of genotype, produced adventitious shoots within four weeks with an average production of 17.5 shoots per embryo. The majority of shoots (on average 77%) were easily converted to whole plants in soil. The white clover regeneration system described is prolific, rapid and effective on a large number of genotypes.Abbreviations BA N⁶-benzylaminopurine - MS medium Murashige & Skoog medium (1962) - NAA -naphthaleneacetic acid - thidiazuron N-phenyl-N-1,2,3-thiadiazol-5-ylurea     

Callus induction and plant regeneration from mature embryos of sunflower

Callus development and efficient shoot and root organogenesis were obtained from five different sunflower (Helianthus annuus L.) genotypes: Trakya 80, Trakya 129, Trakya 259, Trakya 2098, and Viniimk 8931, which are commercially important for Turkey. Plant tissue culture systems were established on Murashige and Skoog (MS) media supplemented with various plant growth regulators using mature embryos of sunflower. For callus induction MS + 1 mg/l 2,4-D, for shoot regeneration MS + 1 mg/l benzyladenine and 0.5 mg/l α-naphthaleneacetic acid were used. Callus induction ratios were around 80–92% in all tested genotypes. The Trakya 259 genotype gave the best shoot regeneration response (44%). All regenerated shoots were rooted on MS medium supplemented with 1 mg/l indolyl-3-butyric acid and on MS medium without any hormones. Mature embryos could be an alternative source for indirect plant regeneration and gene transfer systems for different sunflower genotypes. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 4, pp. 621–624. The text was submitted by the authors in English.     

Plant regeneration and variants from calli derived from immature embryos of diploid barley (Hordeum vulgare L.) and H. vulgare L. x H. bulbosum L. crosses

Summary Plant regeneration from calli was carried out at two locations using several parental genotypes and environments. Selfed immature diploid (VV) barley (Hordeum vulgare) embryos and immature haploid (V) or hybrid (VB) embryos from crosses between H. vulgare and H. bulbosum were used as explants. Golden Promise, Emir and CB 7432 were the best cultivars for plant regeneration, and 15°C tended to be more suitable for plant development than higher temperatures. A total of 844 regenerants were obtained, and over 200 fertile progenies were screened agronomically. Apart from the occurrence of polyploidy and albinism, three variants were identified. One showed maternal inheritance for yellow leaf striping while the other two were controlled by single recessive genes. One of these possessed increased resistance to scald (Rhynchosporium secalis) compared with the donor parent cv Triumph, and one resembled a chlorina mutant.     

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.     

Somatic embryogenesis from immature zygotic embryos of Rosa bourboniana desp

Summary A protocol for the inducton of somatic embryogenesis from immature zygotic embryos of Rosa bourboniana, a scented rose species, was established. Somatic embryos were induced after 8wk of inoculation of zygotic embryos on MS medium supplemented with different concentrations of 2,4-dichlorophenoxy acetic acid (5–15 μM). In addition to 2,4-dichlorophenoxy acetic acid concentrations, somatic embryogenesis was also influenced by the month of collection of the explant and the stage of maturity of the hip. Maximum embryogenic response (16.6%) was observed using 2,4-dichlorophenoxy acetic acid (15 μM), from green hips in the month of September. The use of l-proline (800 mg l⁻¹) was found to be optimum for secondary embryogenesis. On periodic subculturing, the cultures formed somatic embryos sustainably over a period of 18 mo. For somatic embryo germination, 6-benzylaminopurine (5 μM) was found to be most suitable. Rooted plants were transferred successfully to soil and appear morphologically normal under greenhouse conditions. Transfer of plants for hardening was most suitable during the active growth period between June and September. IHBT Publication No: 0447     

Somatic embryogenesis and plant regeneration from immature inflorescence segments of Coix lacryma-jobi

Callus was obtained from segments of immature inflorescence of Coix lacryma-jobi cultured on N6 medium containing 1–2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 3–5% sucrose. Plantlets were regenerated when embryogenic calluses were transferred onto MS medium with 0.5 mg/l kinetin and 0.01 mg/l naphthaleneacetic acid (NAA). Regenerated plants had the diploid chromosome number (2n=20).