Micropropagation of Morus laevigata Wall. from mature trees

Multiple shoots were obtained from nodal explants of 10-year-old tree of Morus laevigata on Murashige and Skoog's medium supplemented with different concentrations (0.5–5.0 mg.l^–1) of benzyladenine (BA). Nodal segments taken from in vitro proliferated shoots gave further multiple shoots when cultured on the same basal medium containing 2.5 mg.l^–1 BA. Repeated subculture resulted in rapid shoot multiplication at the average rate of 6-fold per subculture. In vitro raised shoots rooted on MS medium containing 0.1 mg. l^–1 each of 3-indolebutyric acid (ISA) and -naphthaleneacetic acid (NAA). The regenerated plantlets were successfully established in soil under field conditions after a few days of indoor acclimatization.     

The control of in vitro direct main stem formation of Lilium longiflorum derived from receptacle culture, and rapid propagation by using in vitro stem nodes

The control of in vitro direct main stem formation by culturing receptacles, and a protocol for the micropropagation of Lilium longiflorum using in vitro main stem nodes derived from receptacle culture were developed. Receptacles from flowers cultured on MS medium containing 1.0 mg l^–1 gibberellic acid (GA₃) and 0.5 mg l^–1 6-benzyladenine (BA) resulted in direct main stem formation after 3 months culture. These stems were isolated and cut into nodal stem segments, which were then cultured on MS medium supplemented with 0.2 mg l^–1 BA. Shoots formed on each node after one month culture. These shoots were subcultured on MS medium containing 0.5 mg l^–1 BA for their mass propagation. An average of 30 vigorous and uniform shoots were formed per single shoot after each subculture. A cyclic and continuous system of propagation by multiplication of shoots was developed. Shoots were rooted on 1/2 MS medium containing 0.2 mg l^–1-naphthaleneacetic acid (NAA). One hundred plantlets that were acclimatized in the greenhouse had a 100% survival. A comparison was made with the traditional culture of explants derived from bulb-scales and with that from main stems.     

Regeneration of `juvenile' plants of black plum,Syzygium cuminii Skeels,from nodal exp of mature trees

Nodal explants, excised from young shoots from mature trees of Syzygium cuminii, were cultured on MS medium supplemented with different concentrations of BA or kinetin. Among these, BA (0.5 or 1.0 mg l^–1) induced greening and opening of the incipient shoot buds, which however did not elongate. Elongation of the shoot buds was facilitated on MS medium with 1.0 mg l^–1 BA supplemented with casein hydrolysate (1.5 g l^–1) or glutamine (200 mg l^–1). Nodal explants (microcuttings), taken from shoots developed in vitro, also developed multiple shoots when cultured on MS with1 mg l^–1 BA. These explants did not require an additional supply of reduced nitrogen, for further normal development. Shoots developed from explants from mature trees and microcuttings were rooted by sub-culturing them on Knop's medium supplemented with 2% sucrose and 1 mg l^–1 IAA. The plants that developed in vitro were planted in soil and were transferred to the field after an acclimatization period of 7–8 months. These plants have been thriving well for more than three years and have no apparent phenotypic aberrations.     

Micropropagation of zedoary (<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Roscoe) – a valuable medicinal plant

Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l^–1 sucrose and 5 g l^–1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l^–1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l^–1 BA, and 0.5 mg l^–1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l^–1 sucrose and 8 g l^–1 agar, supplemented with 20 (v/v) CW and 2 mg l^–1 NAA. More than 95 of the rooted plants were established in pots after hardening.     

Plant regeneration from axillary buds of a mature tree of Ficus religiosa

Dormant axillary buds of nodal explants collected from a mature (35-year-old) tree of Ficus religiosa L. sprouted on MS medium supplemented with 6-benzyladenine (BA, 5 mg l^–1) and indole-3-butyric acid (IBA, 0.2 mg l^–1 ) within 4 days. Multiple shoots were obtained when these explants were transferred to MS medium containing 1.5 mg l^–1 BA and 1.5 mg l^–1 adenine sulphate (ADS). These multiple shoots (1 cm) were allowed to elongate to a height of 4–5 cm by maintaining them on MS medium containing 2 mg l^–1 BA, 0.5 mg l^–1 1-naphthalene acetic acid (NAA), and activated charcoal (0.3% w/v). Nodal segments taken from these in vitro-proliferated shoots yielded multiple shoots when cultured on the multiple shoot-inducing MS medium mentioned above. Root induction in these shoots (4–5 cm in height) was achieved by transferring them onto MS medium supplemented with 2 mg l^–1 IBA and 0.1 mg l^–1 NAA for 1 week; upon transfer to half-strength MS basal medium these shoots exhibited root proliferation. These rooted plantlets were successfully established in soil after a short period of acclimatization. Received: 17 April 1997 / Revision received: 2 September 1997 / Accepted: 20 September 1997     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

Efficient GA3-assisted plant regeneration from cell suspensions of three grape genotypes via somatic embryogenesis

Petioles from in vitro grown plants of interspecific grapevine hybrids cvs `Bianca', `Podarok Magaracha' and `Intervitis Magaracha' were cultured on solid NN medium supplemented with 2,4-D and BA at various concentrations. The callus developed was cultured in liquid NN medium supplemented with 0.5 mg l<sup>–1</sup> BA to induce formation of somatic embryos. Somatic embryos of globular and heart-stage developed in suspensions of `Podarok Magaracha' and `Intervitis Magaracha'. In contrast, `Bianca' did not undergo embryogenesis beyond globular stage. This made it necessary to perform subculture of the suspensions to HTE liquid medium supplemented with 0.2 mg l^–1 BA for the development of globular embryos into heart stage. Heart-stage embryos developed into torpedo-stage after subculturing suspensions of all three cultivars to liquid HTE medium supplemented with 0.1 mg l^–1 IAA and 30 mg l^–1 sodium hummate. Torpedo-stage embryo suspensions were subcultured in liquid HTE medium supplemented with 0.5 mg l^–1 BA, 0.5 mg l^–1 GA₃ and 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA. After 12 days of incubation, plantlets were cultured on solid M2MS medium: without growth regulators and with 0.5 mg l^–1 BA. Plantlets that developed in liquid HTE media with 0.5 mg l^–1 GA₃ or 0.5 mg l^–1 GA₃ + 0.2 mg l^–1 BA produced 82–90% shoots on solid M2MS medium with 0.5 mg l^–1 BA after 50 days of culture.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the chinese medicinal plant, <Emphasis Type="Italic">Dendrobium candidum</Emphasis> wall. ex lindl., from axenic nodal segments

Summary Dendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l⁻¹ sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l⁻¹ HYPONeX, 80 g l⁻¹ potato homogenate and 2 g l⁻¹ activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l⁻¹ NAA and the plantlets were successfully acclimatized in soil.     

Rapid plant regeneration through <Emphasis Type="Italic">in vitro</Emphasis> axillary shoot proliferation of butterfly pea (<Emphasis Type="Italic">Clitoria ternatea</Emphasis> L.)—a twinning legume

This study describes a reproducible protocol for rapid mass propagation of a multipurpose legume, Clitoria ternatea L., using cotyledonary node explants derived from axenic seedlings. Multiple shoots were induced in Murashige and Skoog (MS) medium supplemented with N ⁶-benzyladenine (BA), zeatin riboside, or thidiazuron. N ⁶-Benzyladenine at 1.0 mg l⁻¹ (4.44 μM) was most effective for shoot proliferation. Multiple shoots were also induced in nodal segments of in vitro-raised shoots grown on MS medium containing 1.0 mg l⁻¹ (4.44 μM) of BA. Rooting was best induced in shoots grown on half-strength MS medium with 0.25 mg l⁻¹ (1.42 μM) of indole-3-butyric acid. Plants were acclimatized in vermicompost and established in soil where they flowered and formed mature seeds.     

Micropropagation of Spilanthes acmella Murr.

Multiple shoots of Spilanthes acmella Murr. were induced from hypocotyl segments obtained from 1-week-old seedlings on Murashige and Skoog's (MS) medium containing benzyladenine (BA), isopentenyl adenine, and naphthaleneacetic acid (NAA). High frequency shoot proliferation (95 %) and maximum number of shoots per explant (10 ± 0.6) were recorded with 0.5 mg dm^–3 BA in combination with 0.1 mg dm^–3 NAA. A proliferation was achieved by repeatedly subculturing the nodal segments on shoot multiplication medium. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing indole-3-butyric acid (1.0 mg dm^–3). 95 % of the plantlets were successfully acclimatized and established in soil. Transplanted plantlets showed normal flowering without any morphological variation.     

Micropropagation of <Emphasis Type="Italic">Drosophyllum lusitanicum</Emphasis> (Dewy pine), an endangered West Mediterranean endemic insectivorous plant

In this work, in vitro clonal propagation of Drosophyllum lusitanicum (Dewy pine) was obtained from seedlings germinated in vitro. Seeds were collected in various populations identified in the Algarve region and germinated in vitro on MS medium supplemented with 0.5 mg l^–1 BA (6-benzyladenine) and 0.1 mg l^–1 GA₃ (gibberellic acid). The obtained shoots were used in several multiplication assays. The best results were observed in MS medium supplemented with 0.2 or 0.5 mg l^–1 zeatin. The highest rooting frequency (83%) was observed on 1/4MS medium supplemented with 0.2 mg l^–1 IBA (indole-3-butyric acid). Fifty percent of the plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development. Plans are underway to reintroduce the in vitro produced plants from this study in selected locations in their natural habitat.     

Callus induction and high-frequency plant regeneration from hypocotyl and cotyledon explants of Arctium lappa L.

Summary This study describes a protocol for plant regeneration from cultured seedling explants of Arctium lappa. Hypocotyls and cotyledons of A. lappa were induced to form callus by culturing on Murashige and Skoog (MS) medium supplemented with 2.0mg l^&#x2212;1 2,4-dichlorophenoxyacetic acid and 0.5&#x2013;2.0 mg l^&#x2212;1 benzyladenine (BA). Formation of adventitious buds could be induced from calluses or explants directly by culturing on MS medium containing 1.0&#x2013;2.0 mg l^&#x2212;1 &#x03B1;-naphthaleneacetic acid (NAA) and 0.5&#x2013;2.0 mg l^&#x2212;1 BA. These regenerated shoots were rooted on MS medium with 1.0 mg l^&#x2212;1 indole-3-butyric acid or indole-3-acetic acid in combination with 1.0 mgl^&#x2212;1 NAA. The regenerated plants acclimatized in soil were normal morphologically and in growth characters. They flowered and set seeds in the following year after acclimatization.     

Efficient plant regeneration in vitro in buckwheat

An in vitro highly efficient plant regeneration system was established from hypocotyl segments in buckwheat (Fagopyrum esculentum Moench.). Calli were induced on Murashige–Skoog (MS) medium containing 1.0 mg l^–1 to 2.0 mg l^–1 2,4-dichlorophenoxyacetic acid and 1.5 mg l^–1 6-benzylaminopurine. Shoot buds were formed on subcultured pieces of callus. A high frequency (over 80%) of shoot differentiation was obtained on MS medium supplemented with 2.0 mg l^–1 6-benzylaminopurine and 1.0 mg l^–1 6-furfurylaminopurine. The regenerated shoots rooted readily on MS medium plus 0.2 mg l^–1naphthaleneacetic acid and 0.2 mg l^–1 indole butyric acid. The regenerated plantlets were acclimatized and successfully transferred to pots. Chromosome examination showed that the regenerated plants had normal chromosome number (2n=16).     

Plant regeneration from protoplasts isolated from cotyledons of Sesbania bispinosa

Protoplasts were isolated from cotyledons of Sesbania bispinosa (Jacq.) W.F. Wight. In a liquid-over-agar culture system with Murashige and Skoog (MS) medium supplemented with 1 mg l^-1 2,4-dichlorophenoxyacetic acid (2,4-d, 2 mg l^-1 benzyladenine (BA), 1 mg l^-1 glutamine and 0.5 and formed callus. The first division occurred after 3–4 days. Callus formed from the protoplasts differentiated shoots by organogenesis on MS medium with 1 mg l^-1 indolebutyric acid (IBA) and 1 mg l^-1 BA. These shoots developed into complete plantlets when excised and cultured on MS medium with 0.5 mg l^-1 IBA.     

In vitro multiplication ofVanilla planifolia using axillary bud explants

A clonal propagation method has been developed for efficient multiplication ofVanilla planifolia. Multiple shoots were developed from axillary bud explants using semi-solid Murashige and Skoog (MS) medium supplemented with N⁶-benzyladenine (BA, 2 mg l^–1) and -naphthaleneacetic acid (NAA, 1 mg l^–1). The multiple shoots were transferred to agitated liquid MS medium with BA at 1 mg l^–1 and NAA at 0.5 mg l^–1 for 2–3 weeks, and subsequently cultured on semi-solid medium. Using this method, an average of 42 shoots were obtained from a single axillary bud explant over a period of 134 days. Use of an intervening liquid medium has been found to enhance multiplication of shoots inV. planifolia.Abbreviations BA N⁶-benzyladenine - DMRT Duncan's multiple-range test - KC Knudson (1946) medium - KCB KC basal medium - Kn kinetin - MS Murashige and Skoog (1962) medium - MSB MS basal medium - 1/2 MSB half-strength MSB - MS-D double-phase MS medium - MS-L liquid MS medium - MS-S semi-solid MS medium - NAA -Naphthaleneacetic acid     

Effect of genotype,explant type and growth regulators on organogenesis in Morus alba

Plantlets of the mulberry (Morus alba L. vars. Chinese White, and Kokuso-27) were produced from callus cultures. For callus induction, leaf, internodal segments, and petiole explants of Chinese White, Kokuso-27 and Ichinose varieties were grown on MS basal medium fortified with 2,4-D and 6-benzylaminopurine (BA). Callogenesis was dependent on the nature of explant used, the genotype and growth regulators supplemented in the medium. Leaves were the best explant type used for callus induction. Best callogenesis was obtained on MS medium containing a combination of 1 mg l⁻¹ 2,4-D and 0.5 mg l⁻¹ BA (95-100%). Calluses formed shoots on MS medium supplemented with 1 mg l⁻¹ BA. Supplementation with 0.1 mg l⁻¹ 2,3,5-triiodobenzoic acid (TIBA) in this medium enhanced shooting response. Presence of TIBA in the medium also improved the long-term organogenic potential of the callus. Regenerated shoots produced roots on Murashige & Skoog (MS) medium containing either 0.5 mg l⁻¹ indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Seventy percent of the rooted plants were established in the field where they are performing well. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from suspension cultures of Hosta sieboldiana

Systems for establishing suspension cultures and for inducing plant regeneration from these cultures for the Liliaceous ornamental plant, Hosta sieboldiana (Lodd.) Engl. have been developed. Pale-yellow and nodular calluses were induced from more than 20% of scape segments on MS medium containing 1 mg l^–1 picloram (PIC), 30 g l^–1 sucrose, and 2 g l^–1 gellan gum. Upon transfer of calluses to the same medium lacking gellan gum, stably-growing suspension cultures were established after 1 month. Suspension cell clusters regenerated a large number of adventitious shoots following transfer to MS media containing 0.1 mg l^–1 NAA in combination with either BA or TDZ. Over 20 shoots per 0.3 g FW of cell clusters were obtained on media containing 0.1 mg l^–1 NAA and either 1 or 5 mg l^–1 TDZ. Shoots rooted easily on plant growth regulator (PGR)-free MS medium, and plantlets were successfully transferred to soil. Plants showed no visible morphological alterations and maintained the diploid level as indicated by flow cytometric analysis.     

Effects of auxins and cytokinins on formation of Catharanthus roseus G. Don multiple shoots

The effects of different combinations of plant growth regulators and light intensity on the formation of multiple shoots of Catharanthus roseus (L.) were studied. By composing three dimension surfaces and their topo views from experimental data, it was clear that Murashige-Shoog (MS) medium supplemented with 7.0 mg l^-1 BA and 1.0 mg l^-1 NAA strongly stimulated the formation of shoots, whereas medium supplemented with 2,4-d suppressed the formation of shoots or caused shoot dedifferentiated. Light intensities of 550–700 Lux were found to be beneficial to the formation of shoots when MS medium was supplemented with 2 mg l^-1 6-BA and 0–1.0mg l^-1 NAA.Abbreviations BA-6 benzyladenine - NAA -naphthalenacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

Callus induction and plantlet regeneration in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) dunal

Summary Callus induction was observed from hypocotyl, root, and cotyledonary leaf segments, grown on Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KN). Maximum callusing (100%) was obtained from root and cotyledonary leaf segments grown on MS medium supplemented with a combination of 2 mg l⁻¹ (9.1 μM) 2,4-D and 0.2 mg l⁻¹ (0.9 μM) KN. The calluses, when subcultured in the same medium, showed profuse callusing. However, these calluses remained recalcitrant to regenerate regardless of the quality and combinations of plant growth regulators in the nutrient pool. When hypocotyl segments were used as explants, callus induction was noticed in 91% of cultures which showed shoot regeneration on MS medium supplemented with 2 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ KN. These shoots were transferred to fresh medium containing various concentrations and combinations of 6-benzyladenine (BA) and N⁶-(2-isopentenyl)adenosine (2-iP). Maximum shoot multiplication was observed after 60 d of the second subculture on MS medium containing 2 mg l⁻¹ (8.9 μM) BA. These shoots were rooted best (87%) on MS medium containing 2 mg l⁻¹ (9.9 μM) indole-3-butyric acid (IBA). The plantlets were transferred to the field after acclimatization and showed 60% survival.     

Plant regeneration through somatic embryogenesis from callus cultures of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis>

A new method was established for somatic embryogenesis and plant regeneration from callus cultures of Dioscorea zingiberensis C.H. Wright. Primary callus was induced by culturing stems, leaves and petioles on Murashige and Skoog (MS) medium supplemented with 0.5–2.0 mg l^–1 N⁶-benzyladenine (BA) and 0–2.0 mg l^–1 -naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) for 1 month. The highest frequency (87%) of callus formation was achieved from stem explants treated with 0.5 mg l^–1 BA and 2.0 mg l^–1 2,4-D. Somatic embryos were obtained by subculturing embryogenic calli derived from stem explants on MS medium supplemented with 2.0–4.0 mg l^–1 BA and 0–0.4 mg l^–1 NAA or 2,4-D for 3 weeks. The optimum combination of 4.0 mg l^–1 BA and 0.2 mg l^–1 NAA promoted embryo formation on one-third of the calli. After a further month of subculture on the same medium, mature embryos were transferred to MS medium supplemented with 0–4.0 mg l^–1 BA, NAA or indole-3-butyric acid (IBA) for further development of plantlets and tuber formation. Plant growth regulators had a negative effect on the development of mature embryos.