In vitro clonal propagation of tea (Camellia sinensis (L.) O. Kuntze)

A system for in vitro clonal propagation has been developed in tea plants. Shoots obtained from primary explants were induced from terminal buds and axillary buds of mature field-grown plants. Cultures were initiated from both types of explants on Murashige and Skoog (MS) medium supplemented with 10% coconut milk (CM), 200 mg l^-1 of yeast extract (YE), 1.4 M indoleacetic acid (IAA) and 17.8 M benzyladenine (BA). The shoot tips were multiplied on 1/2 strength MS medium containing 10% CM, 2.9 M IAA and 17.8 M BA. The larger shoots were separated after multiplication and rooted on 1/2 MS medium supplemented with 11.4 M ascorbic acid and 34.5 M indolebutyric acid (IBA). A pretreatment of the plants with an aqueous solution of 493 M IBA greatly increased the frequency of rooting. More than 60% of the rooted plants have been transferred to soil successfully.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - YE yeast extract - CM coconut milk - MS Murashige and Skoog medium (1962)     

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     

In vitro organogenesis of elephant apple (Feronia limonia)

Elephant apple (Feronia limonia L.). was micropropagated on MS medium containing 4.4 M benzyladenine and 4.6 M kinetin using cotyledon explants taken from in vitro-grown seedlings. Adventitious buds formed on the cotyledon developed into shoots that were rooted in half-strength MS medium containing 0.57 M indoleacetic acid and 0.49 M indolebutyric acid. Plants were successfully established in soil.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IBA 3-indolebutyric acid - MS Murashige & Skoog     

Regeneration of plantlets through organogenesis in the Himalayan yellow poppy,Meconopsis paniculata

Plantlet formation through organogenesis in callus cultures of Himalayan yellow poppy,Meconopsis paniculata D.Don (Prain), a threatened taxon of ornamental value, is described. Hypocotyl segments from 3-month-old laboratory-raised seedlings produced callus on agar-solidified Murashige and Skoog medium (MS) containing 10 M -naphthaleneacetic acid and 1 M kinetin. Shoots differentiated best from callus on MS containing 10 M indolebutyric acid (IBA) and 1 M 6-benzyladenine. The regenerated shoots rooted best on MS medium containing 10 M IBA. From seed germination to differentiation of plantlets through the two-step organogenesis process required 28–29 weeks.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - FAA formalin-acetic acid-alcohol - BA 6-benzyladenine - IAA indole-acetic acid - IBA indolebutyric acid - GA₃ gibberellic acid - NAA -naphthaleneacetic acid - RH relative humidity     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

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     

Rapid clonal propagation of guava through in vitro shoot proliferation on nodal explants of mature trees

In vitro clonal propagation of guava Banaras local was achieved by culturing nodal explants of mature trees on Murashige and Skoog (MS) revised medium supplemented with 4.5 M 6-benzyladanine (BA) alone or in combination with either 0.6 M indole-3-acetic acid (IAA), 0.5 M indole-3-butyric acid (IBA) or 0.3 M gibberellic acid (GA₃). Multiple shoots were induced to form by enhancement of axillary branching and BA (4.5 M) without any auxin and gibberellin was found to give best shoot multiplication rate. In this medium 3–6 shoots developed on explants collected from field-grown plants and 5–10 shoots developed on explants taken from in vitro proliferated shoots within 12 wk of culture. A prior transfer of shoot clumps to a medium containing a lower concentration of BA (0.5 M) before harvesting of cuttings for rooting allowed rapid extension growth and increased the number of usable shoots per culture. Adventitious rooting occurred after subculturing excised shoots on a medium containing 1/2 strength MS salts, 1.5% sucrose, 1 M each of IBA and -naphtha-leneacetic acid (NAA), and 1 gl^-1 activated charcoal. Regenerated plantlets were successfully established on soil.     

Micropropagation of Withania somnifera from germinating seeds and shoot tips

Shoot multiplication was achieved in vitro from shoot tips of aseptically germinated seedlings of Withania somnifera L. using low concentrations of 6-benzyladenine (BA), viz. 2.2, 4.4 and 8.9 M. Maximum number of shoots were obtained when 2.3 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 2.5 M indolebutyric acid (IBA) was added to medium containing 4.4 M BA during initiation of shoot multiplication, but not when added later. Direct multiple shoot initiation was also obtained from germinating seeds in the presence of BA alone. Rooting was successful in excised shoots grown on growth regulator-free MS medium. Rooted shoots were successfully established in soil in a greenhouse.     

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     

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     

Production of clonal plantlets of Grevillea robusta in in vitro culture via axillary bud activation

A micropropagation method for Grevillea robusta A. Cunn. was developed using explants from mature trees cultured on Woody Plant Medium plus 4.4 M benzyladenine and 0.27 M naphthaleneactic acid (NAA) for shoot proliferation. Each nodal explant produced three to five shoots within 10 to 12 weeks in three successive transfers. Rooting was obtained by dipping the shoots in 0.54 mM NAA solution. Rooted shoots were established well in soil.Abbreviations BA benzyladenine - GA₃ gibberellic acid - IBA indolebutyric acid - NAA naphthaleneactic 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 plant regeneration from callus derived from root explants of Lathyrus sativus

Protocols have been developed for the in vitro production of plants from callus derived from root explants of Lathyrus sativus cv. P-24. Callus and shoot regeneration were achieved only in MS medium supplemented with 10.7 M naphthaleneacetic acid and an increased concentration of kinetin (0.9 M for 14 days to 1.4 M for 18 days) during callusing. The shoots obtained rooted in 1/2 MS supplemented with 0.5 M indolebutyric acid. During the year plants have been regenerated several times. The requirement for growth regulators is very specific and narrow.Abbreviations IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - BA benzyladenine     

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.     

Callus induction and shoot regeneration in Sempervivum tectorum

Leaf and shoot explants of Sempervivum tectorum L., taken from 14- and 30-day-old plants germinated in vitro, have been studied by using Murashige-Skoog and White basal media with cytokinins (benzyladenine, kinetin) and auxins (indoleacetic acid, naphthaleneacetic acid, indolebutyric acid) in various concentrations. Explants taken from 14-day-old plants died but 30-day-old leaves and shoots produced yellow and soft, as well as green and hard calluses on Murashige-Skoog medium with 4.4–8.8 M benzyladenine and 0.57 M indoleacetic acid. Shoot organogenesis was induced from green, hard callus in a medium with 2.2 M benzyladenine plus either 1.1 M indoleacetic acid or 2.5 M indolebutyric acid. Whole plants were grown on Murashige-Skoog medium without plant growth regulators. On the other hand, White medium was not suitable for raising Sempervivum tectorum in vitro.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - MS Murashige-Skoog - NAA napthaleneacetic acid - W White     

Rapid plant regeneration from callus cultures of Plumbago zeylanica

Rapid plant regeneration was achieved in callus cultures derived from leaf and stem explants of Plumbago zeylanica Linn. on MS basal medium supplemented with 4.44 M 6-BA, 1.42 M IAA and 3% (w/v) sucrose. The rate of shoot bud regeneration was positively correlated with the concentration of growth regulators in the nutrient media. The leaf explants were more responsive (82.3%) than the stem explants on medium containing 1.42M IAA in combination with 4.44 M BA. The rate of regeneration was found to maintain the same level for 12 months without loss of vigour. Rooting of the differentiated shoots was achieved in media having 0.57 M IAA with 2% (w/v) sucrose within 10 days of culture. Regenerated plantlets were transferred to soil which grew normally with a survival rate of 90%. This protocol may help in the conservation of the species and selection of variants that may be induced to widen the genetic base of the genus.     

Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas

Techniques for the regeneration of Jatropha curcas L. from various explants have been developed. Regeneration from hypocotyl, petiole and leaf explants was evaluated on a range of concentrations of zeatin, kinetin and N⁶ benzyladenine (BA) either singly or in combination with indole-3-butyric acid (IBA). Higher regeneration from hypocotyl and petiole explants was obtained on BA with IBA than on zeatin- or kinetin-supplemented media. Leaf discs from the third expanding leaf exhibited higher regeneration potential than those from the fourth leaf. Independent of the explant type, direct adventitious shoot bud induction was recorded highest on MS medium with 2.22 M BA and 4.9 M IBA. Although the same BA concentration but with reduced IBA concentration (0.49 M) proved effective in callus mediated regeneration from hypocotyl and leaf explants, the petioles required lower concentrations of the two growth regulators (0.44 M BA and 0.49 M IBA). Regenerated shoots could be rooted on growth regulator-free gelled full-strength MS medium. Following simple hardening procedures, the in vitro-raised plants could be transferred to soil and grown to maturity in the field.Abbreviations BA N⁶ benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog's (1962) medium - NAA -naphthaleneacetic acid     

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)     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Perilla frutescens</Emphasis> from hypocotyl and cotyledon explants

Organogenetic buds were induced from hypocotyl and cotyledon explants of oil crop Perilla frutescens in Murashige and Skoog (MS) medium supplemented with 5.7 M indole-3-acetic acid (IAA) and 8.9 – 13.3 M 6-benzylaminopurine (BA). Shoots were rooted on MS medium with 2.9 M IAA and 1.4 M gibberellic acid (GA3) and the regenerated plants flowered and set seeds normally.     

Effect of genotype,explant and medium onin vitro regeneration of red pepper

In vitro plant regeneration was achieved inCapsicum praetermissum, C. baccatum andC. annuum cvs. G4, Bhiwapuri Sweet pepper, Cayenne pepper and Hybrid pepper. Shoots were induced from hypocotyl, cotyledon and leaf explants on Murashige and Skoog medium supplemented with 5.7 M indoleacetic acid (IAA)+13.3 M benzyladenine (BA); 22 M BA; and 44 M BA. Analysis of variance revealed that the most significant effect on shoot regeneration was due to the explant and it accounted for 56.3% of total variation observed. The genotype x explant effect on regeneration was minor relative to all other 2- and 3-way interactions because leaf explants consistently regenerated more shoots than hypocotyls or cotyledons in all the genotypes and thereby reduced the variation among the genotypes. Explant x medium interaction revealed that 22 M BA was the best growth regulator supplement in regeneration medium for optimal shoot regeneration from leaf explants. Rooting of regenerated shoots was achieved on 5.7 M IAA-containing medium, and the rooting response was better from shoots induced on medium fortified with 5.7 M IAA plus 13.3 M BA. Complete plantlets with diploid chromosome number (2n=2x=24) were transferred to soil and 60–70% of these plantlets survived and grew well.