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     

Micropropagation of ‘Yamatoimo’ Chinese yam (Dioscorea opposita) from immature leaves

A micropropagation system for Yamatoimo Chinese yam (Dioscorea opposita Thunb.) was developed. Immature leaves collected from virus-free plants growing in the greenhouse were cultured on MS medium supplemented with 8.9 M benzyladenine (BA), 3% (w/v) sucrose and 0.8% (w/v) agar. After 2–3 months, multiple buds that were clumps of green-colored bulbous structures including adventitious buds and meristematic regions 2–3 mm in diameter were formed on immature leaves. Transplanting clusters of multiple buds to fresh MS medium supplemented with 0.11 M -naphthaleneacetic acid (NAA), 0.89 M BA and 6% (w/v) sucrose was effective for inducing shoot formation, leading to plantlet formation. After 6 months, a large number of microtubers, about 3–7 mm diameter, were obtained.Abbreviations NAA -naphthaleneacetic acid - BA benzyladenine - MS Murashige and Skoog     

In vitro regeneration through organogenesis of Meconopsis simplicifolia — An endangered ornamental species

Meconopsis simplicifolia (D.Don) Walp. could be propagated by induction of adventitious shoots from callus produced on hypocotyl, cotyledon and rosette leaf explants of 4-month-old seedlings. Callus was initiated on agar-solidified Murashige and Skoog medium supplemented with 1 M kinetin +10 M -naphthaleneacetic acid (NAA). Shoots formed when the callus was subcultured on medium supplemented with kinetin or benzyladenine (BA) in combination with NAA, indoleacetic acid, indolebutyric acid or gibberellic acid. Excised shoots were rooted on medium containing auxin with 10 M NAA producing the best rooting (55%).Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy-acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA -indolebutyric acid - NAA -naphthaleneacetic acid     

In vitro shoot regeneration from leaf explants of Roman Chamomile

Murashige and Skoog (1962) medium supplemented with 1.0 to 4.5 M of BA and 1.0 M of NAA induced adventitious bud formation and shoot development in leaf explants of Roman Chamomile. A higher number of adventitious buds was observed at the proximal end of the explants. Plantlets were replicated and multiplied on MS medium supplemented with 2.25 M of BA and 0.6 M of IAA. Plantlets were rooted on MS medium supplemented with 0.5 M of IBA and successfully weaned in vivo. The plants grew to maturity with high uniformity and no morphological signs of somaclonal variation.     

In vitro production of flowering shoots in ‘German Red’ carnation: effect of uniconazole and gibberellic acid

Summary Callus regenerated near the base of senescing petals of flower bud explants of German Red carnation (Dianthus caryophyllus L.) produced adventitious flowering microshoots on MS-medium containing benzylaminopurine (8.9 M) and naphthaleneacetic acid (2.7 M). When these microshoots were subcultured with some callus, additional adventitious flowering microshoots were produced from the callus. The production of adventitious flowering shoots continued for many subcultures spanning a period of more than two years. Uniconazole (6.9 M) increased the number of adventitious shoots formed by as much as two-fold but decreased shoot length by about 50%. In contrast, GA₃ (2.9 M) decreased adventitious shoot formation and increased shoot length. Regardless of the growth regulator treatment, virtually all of the adventitious shoots produced flower buds. Thus, the growth regulators influenced flowering only indirectly by altering the number of adventitious shoots produced in vitro. These results demonstrate that the flowering habit of the adventitious shoots of German Red carnation is highly persistent and the flowering stimulus continues to be transmitted to the newly formed microshoots through the callus.     

Plant regeneration from nucllar tissues of Aegle marmelos through organogenesis

A protocol for organogenesis from nucellar explants excised from fertilized ovules of immature fruits of Aegle marmelos Corr. was developed. Adventitious buds were initiated on Murashige and Skoog's (MS) medium containing various combinations of 6-benzyladenine (BA), -naphthalene-acetic acid (NAA), 3-indoleacetic acid and gibberellic acid. Medium containing 4.4 m BA and 2.7 M NAA produced the maximum number of adventitious buds per explant. Shoots were elongated by transferring explants with shoot buds to medium with a low concentration of BA (0.44 M). Rooting of in vitro-regenerated shoots was obtained in half-strength MS medium with 4.9 M indole-3-butyric acid. This is the first report of plant regeneration from nucellar explants of A. marmelos.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - GA₃ gibberellic acid     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.)

Immature and mature nonstratified seeds of white ash (Fraxinus americana L.) were dissected transversely and 2/3 of each seed was placed onto agar-solidified Murashige and Skoog medium. Adventitious buds, shoots, and somatic embryos formed on callus, cotyledons, and hypocotyls of the resulting seedlings. Shoot organogenesis was induced on explants cultured on medium with 10 M thidiazuron but not on explants on media with benzyladenine (BA) or isopentenyladenine. Not all seed sources were equally capable of shoot organogenesis and embryogenesis. Atypical of adventitious regeneration of other woody plants, mature seed explants of white ash were more organogenic with shoots that elongated better than explants from immature seeds. Somatic embryogenesis was observed in cultures where mature seeds were first cultured for 4 weeks on a medium containing 10 M adenine 2,4-dichlorophenoxyacetic acid in combination with 0.1 and 1.0 M thidiazuron, followed by transfer to a medium containing 0.05 M 6-benzyladenine and 0.5 M naphthaleneacetic acid. Adventitious shoots and epicotyls from both seedlings and germinated somatic embryos were rooted under intermittent mist and acclimatized to the greenhouse.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - 2iP isopentenyladenine - NAA naphthaleneacetic acid - TDZ thidiazuron-N-phenyl-N-1,2,3-thiadiazol-5-ylurea - WPM woody plant medium     

Shoot organogenesis and plant regeneration in mulberry (Morus bombycis Koidz): Factors influencing morphogenetic potential in callus cultures

Regeneration of multiple shoots via callus induction and organogenesis was achieved in mulberry (Morus bombycis). Pre-soaked internodal explants in 4.4–8.9 M benzyladenine (BA) formed callus on Linsmaier and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (9.05 M), -naphthaleneacetic acid (2.85 M) and BA (2.2 M). Explants soaked for 48 to 72 h in low levels of BA produced loose and nodular callus that showed regeneration ability. Calluses developed adventitious shoot buds within 3–4 weeks on medium containing BA (8.9 M). Fifteen-week-old calluses developed fewer shoot buds than five-week-old calluses, indicating a decrease in morphogenetic potential with increasing duration of callus cultures. Semi-thin section microscopy was used to evaluate incapability of sustained regeneration. Development of normal shoot bud primordia, due to sub-surface reorganisation, was high in young calluses. The decline in the frequency of shoot bud primordia formation with callus ageing is due to reduced cell division activity in epidermal as well as sub-epidermal layers.     

In vitro plant regeneration from leaves and internode sections of sweet cherry cultivars (Prunus avium L.)

Regeneration of adventitious shoots from leaves and, for the first time, from internode sections were compared and optimized for five economically important sweet cherry cultivars, i.e. Schneiders, Sweetheart, Starking Hardy Giant, Kordia and Regina (Prunus avium L.). The influence of basal media, carbon source, combination and dosage of phytohormones, ethylene inhibitor such as silver thiosulfate and a 16 h:8 h light:dark photoperiod versus complete darkness were evaluated. Both, DKW/WPM (1:1) and Quoirin/Lepoivre (QL) basal media stimulated organogenesis more than QL/WPM (1:1), Chee and Pool (CP), Murashige Skoog (MS), Driver and Kuniyuki (DKW) or woody plant (WPM) media did. An induction phase in darkness resulted in lower or zero regeneration rates. The best regeneration efficiencies were generally obtained with thidiazuron in combination with indole-3-butyric-acid. The addition of silver thiosulfate resulted in a similar or reduced regeneration efficiency. Significant genotypic variability in adventitious bud formation was evident for both explant sources, leaf and internode section. Adventitious shoots were obtained from 11% of leaf explants and 50% of internode sections indicating that shoot regeneration from internodes was significantly more efficient than from leaves.     

In vitro clonal propagation of Pisum sativum L.

A system of in vitro clonal propagation has been developed in Pisum sativum L. (cv. Bohatýr). A modified MS-medium supplemented with 20 M 6-benzylaminopurine (BAP) and 0.1 M -naphthaleneacetic acid (NAA) was used to induce multiple shoot formation from shoot apices, axillary buds of the first normal leaf, axillary buds of the first and second primary scales and axillary buds of cotyledons of 4 to 6 day old pea seedlings. Meristem explants maintained a high proliferation ability in each subculture in the course of 20 months of the culture. Regenerated shoots were rooted in the same basal medium containing 5 M NAA. Rooted plants were cultured in hydroponic pots filled with half-strength MS-medium to attain anthesis and seed maturity. The phenotypic uniformity of the regenerants was evaluated. Cytological investigation confirmed the diploid stage (2n=14) of regenerants and their progeny. Histological studies revealed that proliferating shoots originated from axillary and adventitious buds. In vitro propagation is discussed as related to pea breeding.     

In vitro vegetative propagation of Chinese cabbage

Explants from cotyledons, axillary buds, inflorescence stems and flower buds of Brassica campestris ssp. pekinensis (Lour.) Olsson (Chinese cabbage, cv. Wongbok) were cultured on MS medium with growth regulators. Multiple shoots were obtained from cotyledons, axillary buds and flower buds but not from inflorescence stems. Propagation of shoots from cotyledons was more successful than from axillary buds and flower buds. The vegetative propagation rates varied amongst clones derived from cotyledons of the same cultivar and seed lot. The propagation rates of the cotyledon-derived material followed a normal distribution with an average propagation rate of 2.6 shoots per two weeks subculture when cultured on MS media plus 44.4 m benzyladenine (BA) and 14.8 m -indolebutyric acid (IBA). Shoots from three clones were cultured on MS medium with nine different concentrations of BA. The concentration of BA which promoted the highest rate of shoot propagation varied for the three clones and was in the range 44.4 to 177.6 m.     

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N⁶-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 M to 100 M), although, maximum morphogenic response was observed at 10 M concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 M NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog [14] basal medium - NAA naphthaleneacetic acid - TDZ thidiazuron [N-phenyl-N(-1,2,3,-thidiazol-5-yl)-urea]     

Organogenesis and plant regeneration from immature embryos of Rosa hybrida L.

Intact, flowering, rose plants have been regenerated in vitro from excised embryos of crosses between Bridal Pink (the maternal parent) and several pollen parents. Explanted embryonic tissues developed into an organogenic callus which formed adventitious shoots after several months only on a modified half-strength Murashige & Skoog medium containing 1.0 M BA and 0.05 M NAA. These shoots could be separated, grown individually, rooted in a medium with no BA or NAA, with 1.0 M IBA, and transplanted to greenhouse media. Embryos ranging in age from 21 to 35 days post-pollination formed organogenic callus that eventually regenerated adventitious shoots. Histological examination of normally-developing embryos showed that well-defined embryonic axes were beginning to develop at approximately 20–25 days postpollination. Analysis of populations of regenerated plants from different crosses showed differences in flower color, growth habit, peduncle length, and petal number. This system may be useful for irradiation-mutation breeding and/or for the development of transgenic rose plants using Agrobacterium tumefaciens.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IBA 3-indolebutanoic acid - MS Murashige & Skoog - NAA -naphthaleneacetic acid - 2iP N⁶-(2-isopentenyl)adenosine     

Plantlet regeneration from callus initiated from flower buds in the wild species Allium senescens var. minor

Callus regeneration was observed from flower buds of Allium senescens var. minor inoculated in BDS, MS or B5 medium supplemented with 4.4 M benzyladenine alone or in combination with 4.5 M 2,4-dichlorophenoxy-acetic acid (2,4-d), with 2,4-d and kinetin (4.5 M/4.6 M) or with 5.3 M naphthaleneacetic acid. Ovules enlarged initially but the embryogenic tissue degenerated as callus development progressed from the nectar regions of the petals. Shoot buds and leaf primordia developed from the meristematic protuberances that originated from the surface of the callus. BDS medium with 4.5 M 2,4-d and 13.3 M BA was most suitable for shoot multiplication. The regenerated shoots were rooted in respective liquid medium without any growth regulators and successfully transferred to soil with 90% survival rate.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid     

In vitro propagation of Rauwolfia micrantha, a rare medicinal plant

Shoot tip and single node explants from young shoots of 1-year old flowering plants of Rauwolfia micrantha Hook. f. were cultured on Murashige & Skoog (MS) medium variously supplemented with 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). A combination of 13.2 M BA and 2.68 M NAA induced high frequency (77%) formation of up to 3 shoots from each node in 8 weeks. The regeneration of shoot tips from the field-grown plants and in vitro shoots placed horizontally differed. Repeated subculturing of the shoot tips and single nodes at 6-week intervals for over a year in combination of 4.4 M BA and 0.27 M NAA enabled mass multiplication of shoots without any evidence of decline. Rooting of the excised shoots on medium containing 2.6 M NAA was preceded by callus formation. The rooted plants were removed off the callus, hardened off and 80% established in pots. Micropropagated plants displayed uniform morphological, growth, flowering, fruiting and seed germination characteristics.Abbreviations BA 6-benzyladenie - IAA indole-3-acetic acid - IBA indole-3-butyrie acid - 2-ip 2-isopentenyladenine - MS Murashige & Skoog (1962) - NAA -naphthaleneacetic acid     

Micropropagation of the nickel hyperaccumulator,Hybanthus floribundus (Family Violaceae)

A protocol for micropropagation of the nickel hyperaccumulator Hybanthus floribundus (Lindley) F. Muell. (Shrub Violet) is described in this paper. Healthy callus was first produced from stem and leaf explants on a medium containing half strength Murashige and Skoog medium with 5 M N ⁶-benzylaminopurine (BA) and 0.5 M -naphthaleneacetic acid (NAA). Numerous shoots (>20 shoots per callus) were also successfully grown from callus on this medium. The exposure time of shoots to auxin was critical for successful in vitro rooting. Best rooting efficiency was obtained by transferring shoots to auxin medium (100 M indole-3-butyric acid) for 24 h and then to a medium without growth regulators (about 75% of treated shoots produced healthy roots). Importantly, cloned shoots retained their ability to hyperaccumulate nickel.     

In vitro organogenesis from shoot tip,internode, and leaf explants of Ulmus x ‘Pioneer’

Shoot tip explants of the hybrid cultivar Pioneer responded poorly to initial attempts to establish shoot proliferating cultures on Murashige and Skoog (MS) medium containing 2 or 4 µM benzyladenine (BA) with a four week subculture interval. A combination of weekly subcultures and an MS medium containing 2 µM BA produced shoot proliferating cultures sufficient for micropropagation. Shoot organogenesis was obtained when callus derived from internodes of actively elongating shoots was transferred from a primary medium containing various cytokinins to a secondary medium containing MS salts and 10 µM BA. These small shoots elongated when transferred to a medium containing 2.5 µM BA. Adventitious shoots also differentiated on leaf tissue of Pioneer elm. These shoots appeared to differentiate with little if any intervening callus from the margins of leaves of in vitro grown shoots where these leaves touched the medium (MS medium containing 2 µM BA). Tissue cultured shoots from all sources were rooted, acclimated, and transplanted to the greenhouse or field with good success.Salaries and research aupport provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, and The Nursery Crops Research Laboratory. Journal Article No. 23-86.     

Direct shoot organogenesis and plant regeneration from leaves of grape (Vitis spp.)

Adventitious shoots developed from in vitro-grown leaves of Vitis vinifera cultivars Cabernet Sauvignon, French Colombard, Grenache, Thompson Seedless (syn. Sultana) and White Riesling, V. rupestris cv. St. George (syn. du Lot) and V. vinifera × rupestris cv. Ganzin 1. Leaf explants less than 15 mm long were excised from nodal cultures and cultured on Murashige and Skoog or Nitsch and Nitsch-based regeneration media with 0, 1, 2 or 4 mgl^-1 6-benzylaminopurine (BAP). Adventitious shoots developed within 4 weeks at the petiolar stub and occasionally from wounded lamina tissues. Shoot organogenesis occurred only on media containing BAP and at a higher frequency with 2 mgl^-1 than with 1 or 4 mgl^-1. On media containing 2 mgl^-1 BAP, 47, 67, 60, and 42%, respectively, of leaf explants of Cabernet Sauvignon, French Colombard, Thompson Seedless, and White Riesling produced adventitious shoots compared to 14, 14, and 29%, respectively, for Grenache, St. George, and Ganzin 1. Solid culture medium was superior to liquid medium and transfer frequency on solid medium did not affect the regeneration frequency. Further shoot growth was promoted by the transfer of regenerating tissues to fresh regeneration medium. More than 80% of explants initially producing adventitious buds exhibited further shoot growth, developing an average of more than 6 shoots each. Shoots rooted easily and the resulting plants appeared morphologically identical to parent vines.     

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.