Genotypic and media effects on plant regeneration from cotyledon explant cultures of someBrassica species

Conditions were established for efficient plant regeneration from cotyledon explant calli in different cultivars ofBrassica juncea, B. campestris andB. carinata on Murashige & Skoog's (MS) medium supplemented with various combinations of cytokinins and auxins. Regeneration frequency, however, varied with genotype and the different growth hormone combinations in media. Almost in all species, MS medium with zeatin (1.0 mg 1^-1) and IAA (0.1 mg l^-1) was found to be best for shoot organogenesis followed by the ones containing high kinetin (2.0 mg l^-1) and low IAA (0.02 or 0.2 mg l^-1) concentrations. On these media, the cotyledonary explants invariably underwent callusing followed by multiple shoot formation, which could be separated and subcultured for further propagation. Number of shoots per cotyledon explant cultured varied from 0 to as many as 50. InB. juncea andB. campestris, the regeneration frequency declined sharply in the absence of auxin in medium. BAP in combination with NAA yielded no or a reduced number of shoots. Shoot organogenesis also declined with the reduction in photoperiod from continuous light to 16 hours. Shoots were easily rooted during prolonged incubation on the same medium and whole plants were transferred to pots in the greenhouse and grown to maturity.Abbreviations BAP 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - MS medium after Murashige & Skoog [8] - NAA -napthaleneacetic acid - ZEA Zeatin     

Tissue culture and plant propagation of Gomphrena officinalis — a Brazilian medicinal plant

Leaf and stem segments of Gomphrena officinalis originated from aseptically grown seedlings were used to initiate cultures. Callus production was obtained on gelled Murashige & Skoog medium supplemented with 6-benzylaminopurine alone (1.0, 5.0 or 10.0 mgl^-1) or combined with -naphthalene acetic acid (0.1, 0.5 and 1.0 mgl^-1) after 10 to 15 days of culture, and can be transferred to fresh medium every 30 days. The combinations of 5.0 or 10.0 mgl^-1 of 6-benzylaminopurine with 0.1 mgl^-1 of -naphthalene acetic acid were found to be the best for shoot regeneration. Adventitious shoot formation occurred after 50 to 60 days of culture in leaf and internode stem explants. Nodal segments developed actively growing lateral buds after 30 days of culture. Gelled Murashige & Skoog medium containing 10 mgl^-1 of indole-3-butyric acid was considered optimal for the rooting of shoots. Rooted plants transferred to potting soil could be successfully established.Abbreviations BA 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog - NAA -naphthalene acetic acid     

Somatic embryogenesis from leaf explants of Australian fan flower,Scaevola aemula R. Br.

Somatic embryogenesis from leaf explants of Scaevola aemula R. Br. was achieved. Somatic embryos were induced from explants cultured on MS medium supplemented with 0.2 mg/ 2,4-dichlorophenoxyacetic acid and 0.2–0.5 mg/l 6-benzylaminopurine (BAP). Various developmental stages of somatic embryos were found on this medium—from globular embryos to germinated embryos. The transfer of globular embryos to MS medium containing 0.5 mg/l BAP resulted in a high frequency of shoot regeneration. Leaf explants cultured on MS medium containing different combinations of BAP and -naphthaleneacetic acid formed adventitious shoots and roots. Histological examination confirmed the process of somatic embryogenesis. Induction of somatic embryogenesis in Scaevola provides a system for studying embryogenesis in Australian native plants and will facilitate the improvement of these plants using genetic transformation techniques.Abbreviations ABA Abscisic acid - BAP 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA -Naphthaleneacetic acid - PIPES Piperazine-N, N-(2-ethanesulfonic acid) Communicated by R.J. Rose     

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     

Efficient adventitious shoot regeneration and somatic embryogenesis in pea

A rapid and simple method for adventitious shoot regeneration and somatic embryogenesis from immature cotyledon explants of pea (Pisum sativum L.) is described. Cotyledon size and the explant orientation to the medium surface were shown to have a clear effect on shoot regeneration. The highest frequency of shoot regeneration was achieved when the distal end of the greenest cotyledons (7–8 mm in size) were placed in contact with the agar surface. Shoots rooted at a frequency of 80–90% and grew into normal fertile plants. Somatic embryos were induced in cultures of immature cotyledons on modified MS medium containing high levels of -naphthaleneacetic acid (27–215 M) and 2,4-dichlorophenoxyacetic acid (23–181 M). A higher frequency of somatic embryos with a normal morphology were induced using -naphthaleneacetic acid.Abbreviations BA 6-benzyladenine - 2,4-d dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA -naphthaleneacetic acid     

Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoog's basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l^-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l^-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.Abbreviations BAP 6-benzylamino purine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

In vitro regeneration from hypocotyl and seedling cotyledons of tronchuda (Brassica oleracea var. tronchuda Bailey)

Four tronchuda (Brassica oleracea var. tronchuda Bailey) cultivars were tested for their ability to regenerate in vitro on Murashige & Skoog (MS) medium supplemented with 3 different combinations of -naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP). Explants were either axillary bud-free whole cotyledons or hypocotyls from 7-day-old darkgrown seedlings. The ability to regenerate varied by cultivars, explants and the concentration of growth regulators. Hypocotyl explants of all 4 cultivars, and cotyledon explants of 2 cultivars, developed plantlets within 4 weeks. Hypocotyl explants produced more shoots than cotyledons. Cotyledon explants produced more roots than hypocotyls. Best shoot regeneration was on MS medium supplemented with 2 mgl^-1 BAP and 0.1 mgl^-1 NAA. Portuguesa produced the most shoots. Some regenerants varied in leaf shape and phyllotaxy.Abbreviations BAP benzylaminopurine - NAA napththaleneacetic acid - IBA indolebutyric acid     

Agrobacterium-mediated transformation of Arabis gunnisoniana

An efficient method for adventitious shoot regeneration for Arabis drummondii and a transformation protocol for A. gunnisoniana from hypocotyl explants are described. Hypocotyl explants from 7-day-old aseptically grown seedlings were cultured on MS medium containing plant growth regulators (6-benzylaminopurine, 1-phelyl-3- (1,2,3-thiadiazol-5-yl) urea, -naphthaleneacetic acid and 2,4-dichlorophenoxy-acetic acid). After 4 weeks in culture, high frequency of adventitious shoot regeneration was observed. Regenerated shoots were rooted on half-strength MS basal medium supplemented 1% (w/v) sucrose, with or without NAA. This protocol was then used to produce transformed Arabis gunnisoniana plants. A. gunnisoniana hypocotyl explants were co-cultivated with Agrobacterium tumefaciens strain GV3101 harbouring pBJ40. Transgenic shoots were selected on MS 21 medium supplemented with 50 mg l kanamycin. PCR analysis verified the presence of the nptII gene in the plant DNA isolated from kanamycin resistant shoots.     

Adventitious shoot regeneration from cultured petal explants of carnation

Adventitious shoot regeneration was compared among leaf, stem and petal explants of carnation (Dianthus caryophyllus L.) cv. Scania on MS medium containing different concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). High frequency regeneration was obtained only from petal explants on the media containing 5 to 10 M BA with or without 5 M NAA. Among the cytokinins tested, N-2-chloro-4-pyridyl-N-phenylurea and N-1,2,3-thiadiazol-5-yl-N-N-phenylurea were more effective than BA, kinetin, N⁶-2-isopentenyl adenine and zeatin on regeneration from petal explants. Although, high frequency shoot regeneration was obtained from all petal explants harvested from various developmental stages of buds, a significant decrease in regeneration capacity was observed in the explants obtained from fully-opened flowers. High frequency shoot regeneration was also obtained from the petal explants of cvs. Coral. Lena, Nora and White Sim, and an interspecific cultivar Eolo using the method developed in this study.Abbreviations NAA -naphthaleneacetic acid - BA 6-benzyladenine - GA₃ gibberellic acid - 2iP N⁶-2-isopentenyl adenine - KT-30 N-2-chloro-4-pyridyl-N-phenylurea (also called 4PU) - TDZ N-1,2,3-thiadiazol-5-yl-N-phenylurea (also called thidiazuron)     

Shoot regeneration from stem and leaf explants of Dendranthema grandiflora Tzvelev (syn. Chrysanthemum morifolium Ramat.)

Adventitious shoots were regenerated from leaf and stem explants of eleven chrysanthemum cultivars. The optimum medium for both explant types contained Murashige & Skoog basal medium supplemented with 5 M 6-benzylaminopurine and 5 M -napthaleneacetic acid. Generally, stem explants were superior to leaf explants. There were large cultivar differences in shoot regeneration frequency with three cultivars failing to respond over a wide range of hormone combinations. Shoots on stem explants appeared mainly to originate from cortical cells which rapidly divided and ruptured the epidermis. Regenerated shoots could be easily rooted, transferred to glasshouse conditions, and grown to flowering. All regenerated plants had the same morphological characteristics compared to plants derived from nodes.Supported in part by the National Biotechnology Program Research Grants Scheme in collaboration with Calgene Pacific P/L, Victoria, Australia.     

Plant regeneration in Kentucky bluegrass (Poa pratensis L.) via coleoptile tissue cultures

Summary Plant regeneration in Kentucky bluegrass (Poa pratensis L. cv. Touchdown) via culture of seedling tissues was investigated. When coleoptile, leaf, and stem sections of dark-germinated seedlings were cultured on Murashige and Skoog (MS) medium, different types of callus were produced, depending on the expiant source and growth regulator combinations. Only compact-friable callus (type 3) and moderately compact, friable callus (type 2) produced shoots upon subculture. The nonstructured watery callus (type 4) produced roots without shoots. Shoot differentiation from callus tissues was highest when the culture medium contained 0.2 mgL^–1 picloram + 0.01 mgL^–1 -naphthaleneacetic acid (NAA). Calli grown from coleoptiles had higher shoot regeneration frequency (32%) than that obtained from either stem sections (12%) or young leaf tissues (2%) of the same seedlings. Some organogenic callus lines produced exclusively green plants, while others produced albino shoots or a mixture of green and albino shoots. The green plants were multiplied in a medium containing 0.1 mgL^–1 BAP plus either 0.2 mgL^–1 picloram or 0.1 mgL^–1 indole-3-acetic acid (IAA). Over 90% of the cultures in the shoot proliferation medium produced roots in 4 weeks. The rooted plants were successfully established in soil medium and grown in the greenhouse.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - TDZ thidiazuron     

Plantlet regeneration from mature embryos of Juniperus cedrus

A protocol is described for plantlet regeneration using embryonic explants of Juniperus cedrus Webb & Berth. An average of 6 adventitious buds were induced on whole excised embryos cultured for 15 days on Quoirin and LePoivre (QP) half-strength medium supplemented with 5 M N⁶-benzyladenine. For bud development, explants were transferred to phytohormone-free 1/2 QP medium. For shoot elongation, explants were cultured on 1/2 QP with 0.05% activated charcoal and 2% sucrose. Adventitious shoots were rooted successfully in peat-vermiculite-perlite (1:1:1) moistened with 1/4 QP containing 1% sucrose and 5 M -napthaleneacetic acid, pH 5.0. Axillary shoots elongated spontaneously in culture from leaf axils.Abbreviations AC activated charcoal - BA N⁶-benzyladenine - NAA -naphthaleneacetic acid - QP Quoirin & LePoivre - SH Schenk & Hildebrandt     

Culture conditions effecting plant regeneration from cotyledons of Vigna radiata (L.) Wilczek

Conditions for plant regeneration from excised cotyledons of Vigna radiata were studied. Complete plant developed from the uncallused proximal ends of cotyledons on Murashige & Skoog's (MS), Gamborg's (B₅) and C (MS salts + B₅ vitamins) basal media. The basal medium C was found to be best for plant regeneration. Regeneration frequency, however, varied with genotype, size, orientation and age of explant and the different plant growth regulators combination in the medium. Addition of cytokinins induced callusing at the proximal ends of cotyledons followed by multiple shoot formation. Out of 6-benzyl aminopurine (BAP), kinetin (KIN), N (^–2 isopentyl) adenine (2iP) and adenine sulphate (AS), only BAP and KIN were found to be more effective in enhancing the frequency of shoot regeneration. BAP at 1×10^-1M induced maximum (60%) shoot regeneration whereas maximum number of shoots (8 to 9 shoots) per explant was observed with 5×10^-6M BAP. Cotyledons excised from two-day old seedlings were most regenerative. The regenerative response of cotyledons decreased when sliced into two equal parts either longitudinally or transversely. Callusing and organogenic differentiation occured only if the petiolar end of cotyledons was in contact with medium. None of the tested treatments were effective in inducing shoot bud differentiation from subcultured callus. Well developed shoots rooted when incubated on half strength MS, MS and MS basal medium supplemented with IAA (5×10^-6M). The rooted plants were transferred to pots and later established in the field with 60% success.Abbreviations AS adenine sulphate - BAP 6-benzylaminopurine - B₅ medium after Gamborg et al. [6], - C Medium with MS salts + B₅ vitamins - 2iP N (^–2 isopentyl) adenine - IAA indole-3-acetic acid - KIN Kinetin - MS medium after Murashige & Skoog [21] - NAA 1-napthaleneacetic acid     

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     

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     

High efficiency <Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from cotyledon explants of <Emphasis Type="Italic">Incarvillea sinensis</Emphasis>

Summary A simple and effective procedure has been developed for plantlet regeneration from cotyledon-derived callus of the medicinally important herb and ornamental species, Incarvillea sinensis. An average of 18.4 adventitious shoots per explant were obtained from 100% cotyledon explants cultured on half-strength Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ 6-benzylaminopurine for 3 wk, followed by another 4 wk on hormone-free 1/2×MS medium. The cotyledon explants continued to expand and regenerate new shoots upon repeated subculturing onto fresh medium. Most regenerated shoots (66.9%) were rooted on 1/4×MS mediumcontaining 1.0 mg l⁻¹ indole-3-acetic acid, with an average of about 3.8 roots per shoot. Regenerated plants with well developed shoots and roots were successfully acclimatized in soil and were normal phenotypically.     

A method for long-term micropropagation of Phaseolus coccineus L.

A method for long-term plant regeneration of Phaseolus coccineus L, is described. Shoot-tips and cotyledonary nodes cultured on a Murashige and Skoog medium supplemented with N⁶-benzylaminopurine, 10 M, and -naphthaleneacetic acid, 1M, formed multiple bud-shoots. These shoots were transferred to medium containing BAP 1 M, NAA 0.1 M, and gibberellic acid 3 M to promote shoot growth and further shoot multiplication. Rooting was achieved in medium with 11 M indole-3-acetic acid. Rooted plants grew to maturity and were fertile. Cultures have maintained their ability to regenerate plants for more than two years. A sample of 30 regenerated plants (R₀) was tested for chromosome number, all of them being diploid; seven isozymatic systems were electrophpretically analyzed in 82 R₀ regenerated plants. No differences were observed in their electrophoretic patterns in comparison with those shown by seedlings. Histological studies revealed the origin of buds from calluses via organogenesis.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - ADH alcohol dehydrogenase - GOT glutamic-oxaloacetic transaminase - MDH malate dehydrogenase - 6PGD 6-phosphogluconate dehydrogenase - PGI Phosphoglucose isomerase - PGM phosphoglucose mutase - SK shikimate dehydrogenase     

Regeneration of Lycium barbarum L. plants from leaf tissue,callus culture and callus protoplasts

The possibility of plant regeneration from leaf tissue, callus and callus protoplasts of Lycium barbarum L. has been studied. Leaf segments were cultured on B5 medium (Gamborg et al. 1968) containing 1.5 mg/1 6-benzylaminopurine and 0.5 mg/1 -naphthaleneacetic acid. Regeneration of shoots was initiated after 30 days of cultivation. Callus was obtained from leaf and internode tissues on MS medium (Murashige and Skoog 1962) containing 0.4 mg/1 of 2,4dichlorophenoxyacetic acid. Subsequently, callus was successfully subcultured on the same medium with 1 mg/l of 2,4-dichlorophenoxyacetic acid and 0.2 mg/l -naphthaleneacetic acid. Organogenesis in callus culture was obtained in the course of 40 days after transferring to TM-4 (Shahin 1984). Protoplasts were isolated from callus tissue grown in vitro using an enzymatic method. Cell colonies, minicallus formation and organogenesis were obtained. Shoots were rooted on Murashige and Skoog medium containing 0..1 mg/l -naphthaleneacetic acid. Regenerated plants were transferred to soil and were grown to maturity. Regenerated plants carried normal morphological traits.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - Zea zeatin - GA₃ gibberellic acid - MS Murashige and Skoog medium - B5 Gamborg medium     

Plant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis

Using callus derived from immature embryos, regeneration of viable plants was obtained in soybean (Glycine max (L.) Merr.). Depending on the composition of the medium, regeneration occurred via embryogenesis or via organogenesis. Embryogenesis resulted when embryos were plated on Murashige and Skoog (MS) medium containing 43 M -naphthaleneacetic acid. In work with the cultivar Williams 82, the addition of 5.0 M thiamine HCl increased embryogenesis from 33% to 58% of the embryos plated. Addition of 30 M nicotinic acid to the MS medium enhanced embryogenesis further to 76%. Organogenesis was obtained when medium containing 13.3 M 6-benzylaminopurine, 0.2 M and -naphthaleneacetic acid and four times the normal concentration of MS minor salts was used. Histological studies of these cultures confirmed the organogenic and embryogenic nature of the cultures, by demonstrating the formation of shoot buds and somatic embryos, respectively. Similar responses were obtained in all 54 genotypes tested in this manner. The cultures retained the ability to regenerate complete plants for at least 12 months and 12–15 subcultures. Seeds have been obtained from several regenerated plants and when grown in the field these produced normal-appearing fertile plants.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetio acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Shoog (1962) medium - NAA -naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Abscisic acid: a role in shoot enhancement from loblolly pine (Pinus taeda L.) cotyledon explants

Enhancement of shoot regeneration from loblolly pine (Pinus taeda L.) cotyledon explants was studied by addition of abscisic acid (ABA) to Gresshoff-Doy (GD) shoot induction medium containing benzylaminopurine (BA) and naphthaleneacetic acid (NAA). Addition of ABA (10^–7 M) doubled the morphogenic area of cotyledons and increased the fresh weight of cotyledon explants by 40 to 45% after 4 weeks. A 4-week exposure to ABA resulted in a larger morphogenic area per cotyledon than 3, 2, or 1 week(s) respectively. The enhancement by ABA was related to the explant seed source and was not increased by prolonged exposure. Compared to controls, shoot number was enhanced by 31% and 56% with 2 and 4 weeks of ABA (10^–7 M) exposure, respectively. Abscisic acid has a role in enhancing shoot morphogenesis in loblolly pine.Abbreviations ABA Abscisic acid - BA 6-Benzylaminopurine - GD Gresshoff and Doy (1972) nutrient medium - NAA -Naphthaleneacetic acid.