Regeneration of peach plants from callus derived from immature embryos

Summary Peach plants were repeatedly regenerated from immature embryos but not from callus derived from mature embryos. A white, nodular, highly regenerative callus was obtained when friable, primary callus from immature embryos was transferred from medium containing 4.5 M 2,4-dichlorophenoxyacetic acid and 0.44 M benzyladenine (BA) to media containing 0.27 M -naphthaleneacetic acid (NAA) and 2.2 M BA. This callus retained its morphogenetic potential for a minimum of three subcultures. Green nodular callus, that lacked regenerative capacity, was produced from primary callus derived from mature embryos. Maximum regeneration of shoots occurred when highly regenerative callus was transferred to a medium in which the NAA concentration was reduced five times and the BA concentration was increased two times. Regenerated shoots were rooted in the dark on a medium containing 28.5 M indoleacetic acid. Cytogenetic analysis of regenerated plants indicated that all plants were diploid, 2n = 2x = 16. Phenotypic evaluation of regenerated plants, grown under field conditions, is now in progress.     

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid     

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l^-1), myoinositol (100 mg l^-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 M : 0.02 d^-1) and NAA (5.4 M : 0.06 d^-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 M) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 M)+NAA (0.54 M); Zeatin (45.62 M)+NAA (5.37 M) or BA (8.9 M) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 M)+NAA (1.08 M) or BA (13.32 M) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW^-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.     

Auxin activity of phenylacetic acid in tissue culture

The ability of phenylacetic acid (PAA), a naturally occurring auxin, to initiate and support growth of callus and suspension cultures of several species is reported. Callus tissue of tobacco (Nicotiana tabacum L. var. WI-38), initiated and maintained on a medium with 2,4-dichlorophenoxyacetic acid (2,4-D), was transferred to and maintained on media supplemented with 25–500 M PAA as the only plant growth regulator (PGR). Optimal concentrations of PAA were determined for tobacco callus proliferation in the dark (250 M PAA) and with a 16-h light/8-h dark photoperiod (500 M PAA). Tobacco suspension cultures were maintained for over 28 transfers in media containing 20–40 M PAA as the sole PGR. When tobacco callus tissue maintained on PAA-supplemented media for over 18 months was transferred to liquid media containing kinetin, plantlets were regenerated. Callus of sunflower (Helianthus annuus L. var. Russian Mammoth) proliferated on media containing PAA at 5–250 M as the sole PGR. Similar PAA concentrations inhibited normal development and promoted callus formation in tobacco and pea (Pisum sativum L. vars. common, Frogel, and Frimas) epicotyl tissue. PAA as the sole PGR did not support the growth of soybean (Glycine max (L.) Merrill var. Fiskeby) callus or suspension cultures. Chickpea (Cicer arietinum L. var. UC-5) and lentil (Lens culinaris Medic. var. Laird) callus cultures proliferated on media containing 25–500 M PAA, but habituation of the cultures was common. PAA was not toxic to tobacco, chickpea, and lentil tissues at levels as high as 500 M.Paper No. 88514 of the Journal Series of the Idaho Agricultural Experiment Station, Moscow, Idaho, USA.     

Plant development in long-term embryogenic callus lines of Cucurbita pepo

Embryogenic callus derived from pumpkin hypocotyl segments was induced and maintained for 15 years on MS medium supplemented with the auxins IBA (4.9 M), 2, 4-D (4.5 M) or IAA (5.7 M). On induction media continued embryo maturation and development of adult plants typically failed. Therefore, small embryogenic clumps and individually isolated embryos were subcultured two to four times on one of the conversion media: MS supplemented with 1.5% sucrose and (a) no hormone, (b) 2.9 M IAA, (c) 5.7 M IAA, (d) 11.4 M IAA, (e) 12 M IEt, (f) 3.8 M ABA or (g) 2% activated charcoal. The cell line and the kind of auxin used in the induction and maintenance medium, both had a marked influence on the development of plantlets. The best result was achieved with a line that has been induced and maintained for 15 years on MS with IBA. In the IBA line, out of 100 embryos, 77 developed into plantlets on MS medium supplemented with 11.4 M IAA.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - ABA abscisic acid - 2, 4-D 2, 4-di-chlorophenoxyacetic acid - IEt indole-3-etha-nol - MS Murashige and Skoog (1962) medium     

Initiation and culture of potato tuber callus tissue with picloram

Callus cultures of 7 potato cultivars were initiated from tuber tissue and maintained on Gelrite-solidified media with 1–20 M picloram as the only PGR. Ten M picloram was the optimal concentration for callus induction. By 4–6 weeks after explanting, there was sufficient callus produced for subculture to maintenance media which contained 1–20 M picloram as the only PGR. When grown in the dark at 25°C, subcultured callus typically increased 10-fold in wet weight in 4–5 weeks. The callus produced was friable and a light grey to cream color. Callus cultures were used to establish cell suspension cultures. Callus and cell suspension cultures have been maintained for over 2 years on the picloram containing media.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige-Skoog - NAA naphthaleneacetic acid - PGR plant growth regulator Research paper #9053 of the Idaho Agricultural Experiment Station.     

Selective enhancement of Ipomoea batatas Poir. embryogenic and non-embryogenic callus growth and production of embryos in liquid culture

Embryogenic callus cultures of Ipomoea batatas Poir. produce fast growing non-embryogenic material which soon dominates the cultures. Our objective was to selectively enhance the proliferation of the embryogenic fraction. For this, the effect of BAP and 2,4-D concentrations on growth of embryogenic and non-embryogenic callus were studied and consequently, nutrient media for the production and indefinite maintenance of embryogenic callus without embryo formation were defined. Selective proliferation of embryogenic callus was obtained on solid media with 10 M 2,4-D and 1 M BAP and in liquid media with 5 M 2,4-D. Selective proliferation of non-embryogenic callus occurred in liquid medium with 1 M 2,4-D. In embryogenic liquid culture, embryos were produced with 0–2 M 2,4-D. Increasing 2,4-D concentration from 0 to 2 M in these cultures restricted embryo development.Abbreviations 2,4-D = 2,4-dichlorophenoxyacetic acid - BAP = 6-benzylaminopurine     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

The initiation of callus culture ofMichelia champaca for essential oil production

Summary TheMichelia champaca callus was induced from rachises of theM. champaca flowers on 1/2 MS medium containing 3% sugar and 0.9% agar. The medium was also supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP) as the growth regulators. It was observed that the calli ofM. champaca could be induced on media containing 0–50 M 2,4-D and 0.1–1 M BAP. The calli were grown well on media on media containing 0.1–1.0 M BAP and 2,4-D up to 50 M. As 1 M BAP was used, a lower concentration of 2,4-D was associated with a fast initiation of calli, but the culture of these calli turned brown quickly. To the contrary, a higher concentration of 2,4-D led to a slower rate of callus formation and the culture hardly turned brown. The optimum pH for the cell culture was about 5.6 as 1 M of 2,4-D and BAP were present in the medium.     

The in vitro production of an anthocyanin from callus cultures of Oxalis linearis

Callus growth and the production of anthocyanins were sustained on the salts and vitamins of Murashige and Skoog. Callus growth was stimulated at a concentration of 8–32 M -naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-d). Benzyladenine (BA) and zeatin at 8 M inhibited callus growth whereas isopentenyladenine (iP) stimulated callus growth. NAA repressed anthocyanin production with an increase in NAA from 8–32 M. Anthocyanin synthesis was promoted by an increase in 2,4-d from 0.5 to 2 M and decreased thereafter up to a concentration 32 M 2,4-d. A concentration of 8 M BA, thidiazuron and zeatin, respectively stimulated pigment production. Sucrose stimulated callus growth at 60 mM and pigment production at 120–360 mM.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - iP isopentenyladenine - TZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-yl-urea - Bu-HCl Butanol-2N HCl - BAW Butanol-acetic acid-water     

Transformation of Atriplex gmelini plants from callus lines using Agrobacterium tumefaciens

Atriplex gmelini plants were regenerated via organogensis from hypocotyl explants. Callus lines were induced from the hypocotyl explants on Linsmaier and Skoog (LS) medium supplemented with 1 M benzyladenine and 5 M -naphthaleneacetic acid in the dark. Shoots were regenerated from the callus lines on LS medium supplemented with 20 M thidiazuron and 0.1 M -naphthaleneacetic acid under a high-intensity light condition (450 mol m^–2 s^–1). The regenerated shoots were rooted on LS medium without growth regulators to obtain fully developed plants. We succeeded in transforming Atriplex gmelini from callus lines using Agrobacterium tumefaciens.     

The effect of antibiotics on the inhibition of callus induction and plant regeneration from cotyledons of sugarbeet (Beta vulgaris L.)

Callus induction and plantlet regeneration from cotyledonary expiants of sugarbeet was observed utilizing two media formulations, MS and a modified MS termed RVIM both supplemented with 1.0 g/ml BAP as the sole growth regulator. Callus induction was genotype dependent The USDA line 8787 produced the highest response for callus induction followed by Betaseed 4587 and the USDA line C600. This order was conserved on both media formulations. Shoot induction was consistently higher averaging 32% from the RVIM formulation over the 3 genotypes compared to 25% from MS. The antibiotics geneticin, gentamycin, hygromycin, kanamycin and phleomycin were screened with the modified RV system utilizing Betaseed 4587. Callus growth was inhibited by levels of 50 g/ml geneticin, 150 g/ml gentamycin, 10 g/ml hygromycin, 150 g/ml kanamycin and 20 g/ml phleomycin. The results indicate that the concentrations of antibiotics used to inhibit callus induction will be sufficient for use as selectable markers in transformation experiments with Beta vulgaris.Abbreviations B₅ basal medium (Gamborg et al, 1968) - BAP N⁶-Benzylaminopurine - IBA Indole-3-butanoic acid - MS basal medium (Murashige and Skoog 1968) - RVIM modified MS basal medium (Freytag et al, 1988) - MES (2[N-Morpholino] ethanesulfonic acid     

Plant regeneration from embryo-derived callus of oil palm – the effect of exogenous polyamines

Regeneration in oil palm was achieved through somatic embryogenesis/organogenesis from embryo-derived callus. Callus was induced from mature embryos of the cross 281 (D)×18 (P) on modified MS medium supplemented with 2,4-D (113.12 M) and 2-iP (14.76 M). The embryogenic calluses obtained were transferred to Blaydes medium supplemented with 2,4-D (0.045 M) and one of the following growth regulators: TDZ (4.54 M), zeatin riboside (2.85 M), putrescine (1 mM) and spermine (100 M). Secondary somatic embryogenesis was found to occur in media supplemented with polyamines. The efficiency of formation of somatic embryos, secondary somatic embryos and shoot meristemoids were significantly higher in putrescine containing medium. Histological studies were also undertaken.     

Plant regeneration from callus and explants of Sesbania spp.

Root, hypocotyl and cotyledon explants of Sesbania bispinosa, Sesbania cannabina, Sesbania formosa, and Sesbania sesban were cultured on Murashige and Skoog medium with benzyladenine (BA; 2.22, 4.44, 8.88 M) in combination with 2,4-dichlorophenoxyacetic acid (2,4-d; 2.26, 4.52, 9.05 M), indolebutyric acid (IBA; 0.25, 0.49, 4.92 M) or naphthaleneacetic acid (NAA; 2.69, 5.37, 10.74 M). Although all explant types developed some callus, callus occurred earliest and continued to grow fastest with hypocotyls. Media including 2.4-d or NAA gave the fastest growing callus. Callus was subcultured up to 10 times at 20-day intervals and retained a rapid growth rate. Shoots regenerated readily from both hypocotyls or cotyledons but not from roots. Shoot organogenesis was most frequent with IBA (0.25–4.92 M) in combination with BA (4.44–8.88 M) and did not occur with 2,4-d. With each species at least one medium induced shoot differentiation from more than 50 percent of the callus pieces. With one exception, media containing IBA that induced shoot organogenesis on explants also did so in callus, but media containing NAA, even when effective with explants, did not cause differentiation of callus. Shoots that differentiated were excised and cultured on MS medium without growth regulators or with IBA (2.46, 4.92, 9.84 M). Roots developed after 3–8 days on an appropriate rooting medium, often without IBA. Rooted plantlets were transplanted to pots in a greenhouse and developed into normal plants. Suitable media and protocols for initiating and subculturing callus and regenerating whole plants in vitro from callus and explants have thus been established for four species of Sesbania.     

Stepwise decrease of 2,4-D and addition of BA in subculture medium stimulated shoot regeneration and somatic embryogenesis in buffalograss

Plant regeneration of buffalograss `Texoka' was achieved through both somatic embryogenesis and organogenesis by culturing immature male inflorescences collected from field-grown plants. Three passages of subculture for calluses derived from male `Texoka' on medium containing 2.25, 4.5, or 9 M 2,4-D combined with either 0.44 M or 1.32 M BA led to shoot formation via organogenesis. Higher concentrations of 2,4-D (4.5 or 9 M) resulted in higher percentages of embryogenic callus while 2,4-D at 2.25 M generated shoot-producing callus but with a lower percentage of embryogenic callus. Transfer of calluses from medium containing 4.5 M 2,4-D and 0.44 M BA to the somatic embryo initiation medium containing 0.9 M 2,4-D gelled with either 7 g 1^–1 agar or 3 g 1^–1 Gelrite led to the formation of somatic embryos. Somatic embryo initiation medium gelled with 3 g 1^–1 Gelrite led to significantly higher frequency of somatic embryo formation than in medium gelled with 7 g 1^–1 agar. Callus of a female genotype `315' generated under similar treatments did not produce shoots or somatic embryos.     

Rapid clonal propagation of Dioscorea zingiberensis

A protocol was developed for rapid in vitro propagation of Dioscorea zingiberensis Wright using stems as explants. MS medium with the macroelements at half strength and supplemented with 20.0 g l^–1 sucrose and 8.0 g l^–1 agar was used as basal medium. Lateral buds on nodal cuttings grew into shoots within 20 days after culture on basal medium supplemented with 4.4 M 6-benzylaminopurine (BAP) and 1.1 M -naphthalene acetic acid (NAA). The shoots were cut into segments and cultured on medium with 8.9 M BA and 5.4 M NAA for 30 days for callus formation. The callus was cut into pieces and cultured on medium containing 22.2 M BAP and 1.1 M NAA, on which 87.5% of the callus pieces regenerated multiple shoots within 50 days. The shoots were rooted on medium containing 4.9 M indole-3-butyric acid (IBA) for 20 days. Adventitious buds and shoots could be repeatedly formed after the calli were cut into pieces and cultured on the medium containing 8.9 M BAP plus 1.1 M NAA. More than 85% of the regenerated plantlets survived and grew vigorously 1 month after they were transplanted in vermiculite and each plant formed 2–4 microtubers 3 months of transplanting.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

In vitro organogenesis and plant formation in Vigna radiata (L.) Wilczek

In vitro organogenesis was achieved from calluses derived from cotyledon and hypocotyl explants of Vigna radiata on MS medium. Organogenic calluses were induced from both cotyledon and hypocotyl explants excised from 3-day-old seedlings on MS medium containing NAA (1.07 m and BA (2.22 m) and 2,4-D (0.90 m) and BA (2.22 m) combinations respectively. Regeneration of adventitious shoots from cotyledon derived callus was achieved when they were cultured on MS medium supplemented with NAA (1.07 m), BA (8.88 m) and 10% coconut water. Hypocotyl derived calluses produced adventitious shoots when cultured on MS medium fortified with BA (6.66 m), TDZ (2.5 m) and 10% coconut water. Addition of GA at 1.73 m favored maximum 3 elongation of shoots. Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 4.90 m IBA. Rooted plantlets, thus developed were hardened and successfully established in field. Among the different carbohydrates and media tested, 87.64 m sucrose and MS+B5 medium proved best for maximum production of shoots. This protocol produced an average of seven plants per hypocotyl derived callus and 15 plants per cotyledon derived callus over a period of 3 months.     

Effects of the growth regulator 4PU-30 on growth,K+ content,and alkaloid production in tobacco callus cultures

Growth, K⁺ content, and alkaloid production were compared in nonorganogenetic callus cultures ofNicotiana tabacum cv. Burley 21 grown at 25°C in the dark on two different media: a basal medium with 1 M -naphthaleneacetic acid and 1 M kinetin, and one with 1 M -naphthaleneacetic acid and 1 M 4PU-30 (N-(2-chloro-4-pyridyl)-N-phenylurea). These callus tissues behaved differently not only in growth and K⁺ content but also in alkaloid production. In comparison to cultures grown with kinetin, those grown with 4PU-30 showed a significantly higher fresh weight and dry weight and K⁺ content during the growth period studied. The data clearly indicate a positive correlation between K⁺ uptake rate stimulated by 4PU-30 and cell enlargement rate. However, the alkaloid biosynthesis in the callus tissues was activated by the supply of kinetin and diminished by that of 4PU-30. It thus appears that cellular enlargement of meristematic tissue stimulated by 4PU-30 limited alkaloid production.     

In vitro organogenesis and somatic embryogenesis from punctured leaf of Populus nigra × P. maximowiczii

Various explant sources of Populus nigra × P. maximowiczii were used to examine the effects of growth hormones on morphogenesis in vitro. Initial experiments indicated that punctured leaves were superior to non-punctured ones for both callus growth and formation of shoots and roots on MS medium containing various types and concentrations of growth hormones. After 6 weeks in culture, an average of 178 shoots, 129 roots and 3.1 g fresh weight of callus were directly produced from the abaxial side of each punctured leaf. The best combinations of growth hormones for shoot, root and callus proliferation were 0.88 M BA plus 0.05 M 2,4-D, 0.44 M BA plus 2.69 M NAA and 0.44 M BA plus 2.26 M 2,4-D, respectively. Embryoids were also formed on callus derived from punctured leaves. The number of embryoids varied from 0 to 30 per punctured leaf. Adventitious shoots also developed simultaneously with the embryos. Embryoids were removed with a scalpel at the early developmental stages and placed on MS medium lacking growth regulators. Regenerated plantlets were transferred to pots containing vermiculite for normal growth in the greenhouse.