Micropropagation of Frazinus angustifolia from mature and juvenile plant material

Micropropagation of Fraxinus angustifolia Vahl has been successfully achieved both from mature and juvenile plant material using shoot tip and nodal explants. Several basal media supplemented with benzyladenine (BA) and indolebutyric acid (IBA) were tested for shoot proliferation. The most new explants per mature explant (5.3) was obtained on DKW medium plus 4.4 M BA+0.98 M IBA. The most new explants per juvenile explant (5.6) was produced on QL medium plus 8.9 M BA+0.49 M IBA. Rooting was achieved on WPM supplemented with 0.98–4.9 M IBA. Rooted plantlets were transferred to soil and acclimatized with 85% survival.Abbreviations BA benzyladenine - IBA indolebutyric acid - NAA 1-naphthaleneacetic acid     

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     

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.     

Influence of plant growth regulators,basal media and carbohydrate levels on the in vitro development of Pinus ponderosa (Dougl. ex Law.) cotyledon explants

Applications of in vitro screening techniques for Pinus ponderosa resistance to Peridermium harknessii could be beneficial in a tree breeding program. Plant growth regulators, basal media formula and carbohydrate levels were examined to determine the various effects each would have on excised cotyledon growth and development. Proliferating green callus was initiated from cotyledon explants on SH basal medium containing 4.4 M BA:5.4 M NAA and 1% sucrose. Subsequent subculturing onto LS medium supplemented with 44.0 M BA: 5.4 M NAA and 2% sucrose improved callus maintenance. The highest frequency of caulogenesis from cotyledon explants occurred on a modified GD medium containing 44.0 M BA: 0.054 M NAA and 4% glucose. The influence of nitrogen source, osmoticum and medium salt concentrations are discussed relative to callus initiation and caulogenesis.Abbreviations BA 6-benzyladenine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - CD Campbell & Durzan - GD Gresshoff & Doy - LP Le Poivre - LS Linsmaier & Skoog - MC McCown - SH Schenk & Hildebrandt     

Tissue culture and micropropagation of Cuphea ericoides,a potential source of medium-chain fatty acids

Plant rgeneration occurred on leaf-and stem-derived callus of Cuphea ericoides Cham. & Schlechtd obtained in Murashige and Skoog medium supplemented with auxins [indole-3-acetic acid (IAA), -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-d)] plus cytokinins [6-benzyladenine (BA) or kinetin]. These calluses were subcultured and showed vigorous growth. When subcultured on medium containing 2.22 or 4.44 M BA, the calluses showed profuse regeneration of shoots whereas those subcultured on medium supplemented with 2.69 M NAA or 0.226 M 2,4-d produced numerous roots. Isolated shoots rooted on Murashige and Skoog medium lacking growth regulators or containing 0.54 M NAA or 0.49 M indole-3-butyric acid (IBA). Plantlets were acclimatized to greenhouse conditions.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog medium - NAA 1--naphthaleneacetic acid     

In vitro meristem cloning of Eucalyptus tereticornis Sm.

Rapid multiplication of axillary meristems and direct shoot development occurred from nodal explants of mature Eucalyptus tereticornis Sm. with 5.3 M NAA, 1.1 M IAA and 4.4 M BA in Murashige-Skoog medium. Repeated subcultures of the second generation shoot cultures into low cytokinin-auxin containing media (0.44–0.88 M BA+0.1 M NAA) yielded axillary microshoots in large numbers. Half-strength MS liquid medium with 4.9 M IBA, 5.5 M IAA and 5.3 M NAA for four days, half-strength semi-solid hormonefree MS medium with charcoal, and MS liquid medium without charcoal and hormones, in sequence, induced rooting of shoots in the dark. This system is suitable for the mass propagation of this difficult-to-root eucalypt.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - IBA -indole-3-butyric acid - 2-iP isopentyl adenine - Kn kinetin - MS Murashige-Skoog - NAA -naphthalene acetic acid - PVP polyvinylpyrrolidone     

Whole plant regeneration of Cicer arietinum from callus cultures via organogenesis

A protocol for whole plant regeneration of Cicer arietinum L. cv. C-235 via organogenesis from callus has been developed. Callus initiation was best when immature leaflets were cultured on MS medium containing 5 or 25 M 2,4-D or NAA in combination with 10 M BA, or 25 M 2,4-D alone. The callus grew most vigorously on MS medum supplemented with 10 M NAA and 5 MBA. Best shoot differentiation was obtained from calli derived from the basal portion of shoot tips on MS medium supplemented with 10 M BA and 0.1 M IBA. The shoot forming ability of calli was enhanced by adding 5 mM potassium phosphate to the medium. Shoots were rooted on a MS medium containing l M IBA. The regenerated plants were grown to maturity and produced viable seed.Abbreviations 2,4-D 2,4-dichlorophen-oxyacetic acid - NAA 1-naphthaleneacetic acid - BA benzyladenine - Kn kinetin - 2-ip 6-(,-dimethylallylamino)-purine - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium     

In vitro micropropagation from nodal segments of Cleistanthus collinus

Cleistanthus collinus Benth. was micropropagated using nodal explants on MS medium supplemented with 2.2 M benzyladenine (BA). April to June was the best time for initiating shoot cultures. Shoot proliferation was enhanced when the BA concentration was lowered to 1.1 M. Rooting was achieved on half-strenth MS medium with 22.8 M indole-3-acetic acid for 7 days and continuous darkness for the first 72 h of the 7 days.Abbreviations MS Murashige & Skoog's medium - WPM Woody Plant Medium - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA 1-naphthalenacetic acid     

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     

Adventitious shoot organogenesis and plant regeneration from cotyledons of Dalbergia sissoo Roxb., a timber yielding tree legume

A procedure is outlined to induce adventitious shoot organogenesis from semi-mature as well as mature cotyledons lacking the embryonic axis of Dalbergia sissoo Roxb., an economically important leguminous tree. Shoot buds were induced in the proximal region of the semi-mature cotyledons on Murashige and Skoog's (MS) medium supplemented with 4.44 M 6-benzyladenine (BA) and 0.26 M -naphthaleneacetic acid (NAA). These buds elongated into shoots following transfer to a similar medium containing half-strength macro-nutrients. Adventitious shoot bud formation was also induced in the mature cotyledons. However, unlike the semi-mature explants, the mature cotyledons exhibited shoot bud differentiation on MS medium containing 22.20 M BA without NAA. Pre-culture of mature cotyledons in liquid MS medium containing 8.88 M BA for a duration of 48 h improved shoot bud regeneration up to six-fold. Regenerated shoots, derived from semi-mature and mature cotyledons, rooted on half-strength MS medium containing 1.23 M and 4.92 M indole-3-butyric acid (IBA), respectively. Plantlets were acclimatized and established in soil.     

Organogenesis versus embryogenesis from long-term suspension cultures of cucumber (Cucumis sativus L.)

Suspension cultures were initiated from leaf explant-derived callus of cucumber,Cucumis sativus cv. Hokus, and maintained under two different conditions; (I) continuously in medium with 5 M 2,4-D + 5 M BA, and (II) alternately three cultures in medium containing 5 M NAA + 5 M BA and one culture in 5 M 2,4-D + 5 M BA. After plating on solid medium with 0.5 M KIN + 0.1 M IAA, suspension aggregates from long-term culture in medium with 2,4-D developed into callus, and subsequently formed somatic embryos. These embryos, however, hardly developed into plants. They showed growth arrest and several structural abnormalities. In contrast, organogenesis took place when suspension aggregates from NAA containing medium were plated on solid medium with 0.5 M KIN + 0.1 M IAA. Numerous adventitious buds were regenerated, which quite normally developed into plants. Sucrose at low concentration of 1% improved plant formation. On the average thirty complete plants were obtained from each ml of suspension. It is discussed why adventitious buds develop into plants so well, whereas somatic embryos are prone to growth arrest and abnormal development.Abbreviations BA 6-benzylaminopurine - KIN kinetin - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Organogenesis and somatic embryogenesis in Cupressus sempervirens

Adventitious buds of Cupressus sempervirens L., were formed on excised mature embryos cultured for 10 days on half-strength Quoirin and Lepoivre medium (1/2QP) with 10 M N⁶-benzyladenine. For shoot development, embryos were transferred to 1/2QP without growth regulators. Axillary shoot formation and rooting occurred spontaneously as adventitious shoots aged and transfer intervals were increased. Embryogenic tissue was obtained from immature embryos on induction media consisting of von Arnold and Eriksson (AE) or Gupta and Durzan (DCR) salts with 10 or 20 M 2,4-dichlorophenoxyacetic acid. Cultures were maintained on DCR with 5 M -naphthaleneacetic acid and 5 M BA.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - ABA absicic acid - AC activated charcoal - NAA -naphthaleneacetic acid - QP Quoirin & Lepoivre (1977) - AE von Arnold & Eriksson (1981) - DCR Gupta & Durzan (1985)     

Effects of source of macronutrients and plant growth regulator concentrations on shoot proliferation of Cornus florida

Nodal stem segments of flowering dogwood (Cornus florida L.) were cultured on media containing seven different sources of macronutrients including full- and half-strength Murashige & Skoog (MS) macrosalts, N6, Anderson's (AND), Quoirin & Lepoivre's (LP), Nitsch & Nitsch (NIT), and Woody Plant Medium (WPM). All media contained MS micronutrients, Staba vitamins, 20 g l^-1 sucrose, and 6.5 g l^-1 Difco Bacto-agar, and were supplemented with 2.2 M 6-benzyladenine (BA) and 0.49 M indole-3-butyric acid (IBA). Following three subcultures, the best shoot proliferation was supported on media containing WPM macronutrients. To optimize the proliferation rate, shoots were cultured on WPM macronutrients supplemented with eight combinations of BA and IBA, and 3.3 M BA without IBA was determined to be the best.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid     

Clonal propagation of Camptotheca acuminata through shoot bud culture

The chinese tree Camptotheca acuminata produces the anti-cancer and anti-retroviral drug camptothecin. Methods were developed for the clonal propagation of this important medicinal plant through shoot bud culture. Shoot buds were excised from 25 to 30 day old seedlings, presoaked for 48 h in three different liquid media containing either BA (2.22–17.4 M), kinetin (2.32–18.58 M), or thidiazuron (0.1–10 M) and were subsequently cultured on semi-solid medium of the same composition. Multiple shoots only developed from the 6-benzyladenine presoaked explants with the maximum number of shoots initiated from buds presoaked in and grown on B5 medium containing 17.4 M 6-benzyladenine. Individual shoots were removed from clusters and rooted on B5 supplemented with indole-3-butyric acid (4.9–19.6 M). The lowest concentration of indole-3-butyric acid (4.9 M) gave the highest percentage of rooting (82%) and the shortest root initiation period (18 d). Over 90% of the in vitro rooted plantlets survived transfer to soil.Abbreviations BA 6-benzyladenine - B5 Gamborg's B5 medium (Gamborg et al., 1968) - CPT camptothecin - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - LS Linsmaier & Skoog medium (Linsmaier & Skoog, 1965) - MS Murashige & Skoog (Murashige & Skoog, 1962) - NAA I-naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - WPM woody plant medium (Lloyd & McCown, 1981)     

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     

Plant regeneration from cell suspension cultures of Vigna aconitifolia

Plant regeneration has been achieved routinely from established cell suspension culture lines of Vigna aconitifolia (moth bean), a highly drought tolerant grain legume. The cultures originated from three-week-old leaf callus. Several media including MS, B₅, AA, SL, PCM, SH and L-6 were tested for their effects on cell growth. Maximum growth was observed in L-6 medium containing 44.5 M 2,4-D. After 6 to 8 weeks the suspensions were filtered through 500, 250, 125 and 60 m sieves, respectively, for four to five subcultures. An embryogenic cell line (VA-686) was obtained from the cell fraction collected below 250 m. The VA-686 cell line is being maintained on L-6 medium with 4.5 M 2,4-D and 2.3 M Zeatin. Somatic embryogenesis was induced by transferring the cells to L-6 medium with 4.6 M zeatin in which green cell clusters were produced. The somatic embryos developed from most of the cell clusters when plated on L-6 agar medium with 2.3 M BA.Plantlets were obtained from the embryos on L-6 medium with 10.0 M IBA. The regenerated plants were grown to maturity in the greenhouse.Abbreviations BA Benzyladenine - 2,4-D 2,4- dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IBA Indole-3-butyric acid - IPA Isopentenyladenine - KN Kinetin - NAA Napthaleneacetic acid - AA Toriyama and Hinata, 1985 - SL Phillips and Collin, 1980 A project sponsored by United States Agency for International Development, Washington D.C.     

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     

In vitro clonal propagation of Capsicum spp.

The effect of various concentrations of benzyladenine (BA 4.4–177.5 M) or kinetin (4.7–185.9 M) on shoot proliferation from shoot-tip explants was investigated in C. praetermissum Heiser & Smith and C. annuum L. Maximum number of shoots were obtained on Murashige & Skoog's medium with 66.6 M BA or 92.9 M kinetin in C. praetermissum, and 88.8 M BA or 116.2 M kinetin in C. annuum after 4 weeks of culture. Combining 1 M 2, 3, 5-triiodobenzoic acid (TIBA) with low levels of BA or kinetin significantly increased shoot number as compared to using either cytokinin alone. Rooting of regenerated shoots was achieved on MS medium containing 5.7 M indoleacetic acid. Best rooting (80–100%) was observed in shoots from TIBA plus BA or kinetin media while only 40–50% of shoots from the BA or kinetin treatments were rootable. Plantlets obtained from TIBA plus BA or kinetin were normal diploids while those from BA or kinetin alone revealed distinct chromosomal aberrations in their root tip squashes. Regenerants from TIBA plus BA or kinetin media were successfully established in the soil (86% survival rate), where they flowered and showed normal meiotic behaviour with 100% pollen viability.Abbreviations BA benzyladenine - IAA indole-3-acetic acid - MS Murashige & Skoog's medium - TIBA 2, 3, 5-triiodobenzoic acid     

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)     

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.