Micropropagation of Coleus blumei from nodal segments and shoot tips

A rapid and highly-effective method for micropropagation from nodal segment and shoot tip explants was established for Coleus blumei Benth. Nodal segments and shoot tips were inoculated on MS medium containing 0.7 % agar, 3 % commercial sugar, and different combinations of 6-benzyladenine (BA) with indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthaleneacetic acid (NAA). Hundred percent shoot induction from both explants was achieved on the medium containing BA (2 mg dm⁻³) and NAA (1 mg dm⁻³). Shoot tips were proved to be the better explant in comparison to nodal segments in having high rate of shoot induction and more number of shoots. The same media conditions were found suitable for shoot multiplication. Multiplied shoots rooted best on MS medium supplemented with IBA (2 mg dm⁻³). Micropropagated plants were successfully established in soil after hardening, with 100 % survival rate.     

High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel plant

A simple, high-frequency and reproducible protocol for induction of adventitious shoot buds and plant regeneration from leaf-disc cultures of Jatropha curcas L. has been developed. Adventitious shoot buds were induced from very young leaf explants of in vitro germinated seedlings as well as mature field-grown plants cultured on Murashige and Skoog’s (MS) medium supplemented with thidiazuron (TDZ) (2.27 μM), 6-benzylaminopurine (BA) (2.22 μM) and indole-3-butyric acid (IBA) (0.49 μM). The presence of TDZ in the induction medium has greater influence on the induction of adventitious shoot buds, whereas BA in the absence of TDZ promoted callus induction rather than shoot buds. Induced shoot buds were multiplied and elongated into shoots following transfer to the MS medium supplemented with BA (4.44 μM), kinetin (Kn) (2.33 μM), indole-3-acetic acid (IAA) (1.43 μM), and gibberellic acid (GA₃) (0.72 μM). Well-developed shoots were rooted on MS medium supplemented with IBA (0.5 μM) after 30 days. Regenerated plants after 2 months of acclimatization were successfully transferred to the field without visible morphological variation. This protocol might find use in mass production of true-to-type plants and in production of transgenic plants through Agrobacterium/biolistic-mediated transformation.     

In vitro induction of multiple shoots and plant regeneration from shoot tips of mung bean (Vigna radiata (L.) Wilczek)

Conditions for plant regeneration from excised shoot tips of Vigna radiata were studied. Complete plants were regenerated directly without an intervening callus phase from shoot tips on basal medium (MS salts+B₅vitamins). Regeneration frequency varied with genotype, explant size and growth regulator combinations in the medium. Addition of cytokinins induced a variable amount of callus at the base of the shoot tip, followed by multiple shoot formation. Benzyladenine (BA), kinetin and zeatin at 5×10^-6 M each induced multiple shoots in 100% of the explants but the highest number of regenerants per explant (9) was produced with BA. The efficacy of BA for shoot multiplication was not improved when it was supplemented with naphthaleneacetic acid (NAA) or indoleacetic acid (IAA). NAA or adenine sulphate, when applied alone, induced complete plantlets. The growth regulator requirement of explants for the induction of multiple shoots varied with explant size. The shoot tip explants maintained proliferation ability on subculture. None of the treatments was effective in inducing shoot bud differentiation from callus. Regenerated shoots were rooted on MS basal medium and MS supplemented with either IAA or indolebutyric acid. The rooted plants were transferred to the field; 60% subsequently survived and grew.Abbreviations BM basal medium [MS (Murashige & Skoog 1962) salts+B₅ (Gamborg et al. 1968) vitamins] - BA 6-benzyladenine - AdS adenine sulphate - IAA indole-3-acetic acid - NAA-1 naphthaleneacetic acid - IBA indolebutyric acid     

Micropropagation and acclimatization of Hedeoma multiflorum

A method for the micropropagation of Hedeoma multiflorum Benth from shoot tips or nodal segments was developed. Proliferating microshoot cultures were obtained by placing shoot tips or nodal segments on half-strength Murashige and Skoog (1962) medium supplemented with 22.2 μM BA or 22.2 μM BA plus 0.05 μM NAA. Individual shoots were excised and transferred into rooting medium containing auxins (IBA, NAA or IAA). Rooting of shoots was better on half-strength MS medium containing 0.6 μM IAA than on half-strength MS medium without growth regulators. Rooted plantlets were successfully acclimatized to soil. Preconditioning at different sucrose concentrations prior to acclimatization had no effect on plant establishment, but influenced plant quality. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of Lippia junelliana (Mold.) Tronc.

A method for the micropropagation of Lippia junelliana (Mold.) Tronc. from shoot tips or nodal segments was developed. Proliferating microshoot cultures were obtained by placing shoot tips or nodal segments on full strength Murashige and Skoog medium (MS) supplemented with 4.4 μM benzyladenine (BA) or 0.04 μM indolebutyric acid- (IBA) plus 4.4 μM BA. The rooting of shoots was better on full-strength MS medium without growth regulators. Rooted plantlets were successfully acclimatized to soil. The shoot cultures showed a lower essential oil accumulation in comparison with parent plants. Essential oil accumulation is closely related with growth and shows a negative correlation with shoot proliferation. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro plant regeneration from seedling explants of Stereospermum personatum D.C.: a medicinal tree

Padar (Stereospermum personatum, family Bignoniaceae) is a well-known medicinal tree. Its complete regeneration occurred through shoot bud culture in vitro. The seeds germinated sequentially on plastic trays and polyethylene bags for 21 days served as explants source. Nodal segments from the seedlings were established on MS medium supplemented with 4.44 μM BA, in which 86.6% nodes showed shoot bud elongation. Then, nodal segments from the developed shoots were cultured on MS medium with several BA concentrations; best shoot multiplication was obtained with 0.44 μM BA. In a second experiment where PVP was added to proliferation medium, nodal segments from developed shoots produced maximum 2.78 shoots per node. The nodal segments showed shoot multiplication up to seventh subculture on. Finally, shoots were rooted on MS medium with 2.46 μM IBA. The plants transferred to net pots containing coco-peat were acclimatized in green house, where more than 80% plants survived and grew normally.     

Micropropagation of Bixa orellana using phytohormones and triacontanol

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM TRIA. The primary shoots elongated best on MS medium containing 6.66 μM BA and 2.45 μM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 μM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.     

Micropropagation of <Emphasis Type="Italic">Metrosideros excelsa</Emphasis>

Multiple shoots were induced on stem segments of an 8-y-old plant of Metrosideros excelsa Sol ex Gaertn. “Parnel”. Axillary shoots produced on uncontaminated explants were excised, segmented, and recultured in the same medium to increase the stock of shoot cultures. The Murashige and Skoog (MS) medium, augmented with different concentrations of 2- isopenthenyladenine (2iP) and indole-3-acetic acid (IAA), either singly or in combinations, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment, equal molar concentrations of four cytokinins [2iP, kinetin, zeatin, and N ⁶-benzyladenine (BA)] in combination with equal molar concentrations of three auxins [IAA, α-naphthaleneacetic acid (NAA), and indole-3-butyric acid (IBA)] were tested for ability to induce axillary shoot development from single-node stem segments. The highest rate of axillary shoot proliferation was induced on MS agar medium supplemented with 1.96μM 2iP and 1.14μM IAA after 6 wk in culture. Different auxins (IAA, IBA, and NAA) were tested to determine the optimum conditions for in vitro rooting of microshoots. The best results were accomplished with IAA at 5.71μM (89% rooting) and with IBA at 2.85 or 5.71μM (86% and 86% rooting, respectively). Seventy and 90 percent of the microshoots were rooted ex vitro in bottom-heated bench (22 ± 2°C) after 2 and 4 wk, respectively. In vitro and ex vitro rooted plantlets were successfully established in soil.     

The Effect of Different Plant Growth Regulators on Adventitious Shoot Formation from Virginia Pine (Pinus virginiana) Zygotic Embryo Explants

The effects of different plant growth regulators on in vitro adventitious shoot formation in Virginia pine (Pinus virginiana Mill.) zygotic embryo explants were quantitatively evaluated. Using Tang and Ouyang (1999) (TE) basal medium supplemented with 11.4 μM indole-3-acetic acid (IAA) and 2.2 μM N⁶-benzyladenine (BA), callus was observed after 3–6 weeks of culture. Calluses were transferred to TE basal medium supplemented with 0.49 μM indole-3-butyric acid (IBA) and 8.8 μM BA for 6–9 weeks, where they produced numerous small shoot primordia. They were then transferred to TE basal medium supplemented with 0.49 μM IBA and 4.4 μM BA to promote growth and elongation of adventitious shoots. After elongated shoots were transferred to TE medium containing 0.05 μM α-naphthaleneacetic acid (NAA) for 6 weeks, adventitious roots were formed. Regenerated plantlets were established in soil in greenhouse.     

In vitro propagation of a multipurpose leguminous tree (Pterocarpus marsupium Roxb.) using nodal explants

Pterocarpus marsupium (Bijasal) is a valuable multipurpose forest tree. The regeneration rate in natural habitat is low and the tree is overexploited. An in vitro propagation protocol has been developed from nodal explants obtained from in vitro raised 18-day-old axenic seedlings. The highest shoot regeneration frequency (85%), maximum number of multiple shoots (8.6) as well as length (4.8 cm) were induced from nodal explants on Murashige and Skoog (MS) medium amended with 4.0 μM 6-benzyladenine (BA), 0.5 μM indole-3-acetic acid (IAA) and 20 μM adenine sulphate (AdS). The percentage of shoot multiplication as well as the number of shoots per node remained the same during the first two subculture, afterwards a decline was recorded. Rooting was best induced in microshoots excised from proliferated shoot cultures on semisolid hormone-free half-strength MS medium, after a pulse (dip) treatment for 7 days in half-strength MS liquid medium containing 100 μM indole-3-butyric acid (IBA) and 15.84 μM phloroglucinol (PG). The in vitro-raised plantlets were potted and acclimatized under culture room conditions for 4 weeks before their transfer to a greenhouse, where the established plants showed 75% survival.     

In vitro propagation of Cassia angustifolia through leaflet and cotyledon derived calli

High efficiency shoot regeneration was achieved through leaflet and cotyledon derived calli in Cassia angustifolia - an important medicinal plant. Dark brown compact callus was induced at the cut ends of the explants on Murashige and Skoog's (MS) medium augmented with 1 μM N⁶-benzyladenine (BA) + 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Such callus pieces on transfer to cytokinins (BA or kinetin) supplemented medium differentiated shoots within 10 – 15 d. Of the two cytokinins, 5 μM BA was optimum for eliciting morphogenic response in 83.33 and 70.83 % cultures with an average of 4.16 ± 0.47 and 3.70 ± 0.56 shoots in cotyledon and leaflet derived calli, respectively. The addition of 0.5 μM α-naphthaleneacetic acid (NAA) to MS + 5 μM BA further elevated the maximum average number of shoots to 12.08 ± 1.04 and 5.37 ± 0.52 for cotyledon and leaflet calli, respectively. The excised shoots were transferred to a rooting medium containing either IAA (indole-3-acetic acid), IBA (indole-3-butyric acid) or NAA. Nearly 95 % shoots developed an average of 5.4 ± 0.41 roots on half strength MS medium supplemented with 10 μM IBA.     

Organogenesis, shoot regeneration, and flowering response of Vernonia cinerea to different auxin/cytokinin combinations

Summary A viable protocol has been developed for direct and indirect shoot regeneration of Vernonia cinerea. To establish a stable and high-frequency plant regeneration system, leaf and stem explants were tested with different combinations of α-naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), and benzylaminopurine (BA). Lateral buds on nodal explants grew into shoots within 2 wk of culture in Murashige and Skoog (MS) basal medium supplemented with 20.9 μM BA. Excision and culture of nodal segments from in vitro-raised shoots on fresh medium with the same concentration of BA facilitated development of more than 15 shoots per node. Similarly leaf, nodal, and internodal explants were cultured on MS basal medium supplemented with different concentrations of BA, NAA, and IAA either alone or in combinations for callus induction and organogenesis. Shoot buds and/or roots were regenerated on callus. Shoot buds formed multiple shoots within 4 wk after incubation in induction medium. Adventitious buds and shoots proliferated when callus was cut into pieces and subcultured on MS basal medium containing 20.9 μM BA and 5.3 μM NAA. This combination proved to be the best medium for enhanced adventitious shoot bud multiplication, generating a maximum of 50 shoots in 4 wk. This medium was also used successfully for shoot proliferation in liquid medium. Root formation was observed from callus induced in medium containing 8.05–13.4 μM NAA. Regenerated shoots exhibited flowering and root formation in MS basal medium without any growth regulators. Plantlets established in the field showed 85% survival and exhibited identical morphological characteristics as the donor plant.     

In vitro micropropagation of the tropical fruit tree Syzygium cuminii L.

Multiple shoots were obtained from nodal and shoot tip segments of 10 to 15-day-old seedlings of Syzygium cuminii L. on Murashige & Skoog (MS) revised medium supplemented with 6-benzyladenine (BA) (0.23–8.90 M) singly or in combination with -naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). Excised shoots were placed for root induction on MS medium containing NAA and/or IBA and then transferred to MS basal medium to form complete plantlets. The regenerated plantlets have been acclimatized and successfully transferred into the soil.     

Assessment of factors affecting micropropagation and ex vitro acclimatization of Nyctanthes arbor-tristis L

Rapid differentiation of multiple shoots was observed in 94% of nodal explants of one year old Nyctanthes arbor-tristis L. plants. Shoot bud induction and multiplication took place on Murashige and Skoog (MS) medium supplemented with two cytokinins, i.e. Benzyladenine (BA) or Kinetin (Kn) either alone or in combination with different auxins, indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). Between different media, pH levels and growth regulators tried, the optimum condition for maximum regenerative response was obtained on MS + Kn (2.5 μM) + N AA (0.5 μM) media at 5.8 pH, forming cultures with 23.26 ± 0.89 number of shoots and 6.36 ± 0.80 cm shoot length after 8 weeks of culture. Histological sections confirmed the formation of multiple buds from nodal explants. Rooting was achieved ex vitro by dipping the basal ends of microshoots in 200 μM IBA for 30 min followed by their transplantation in sterile soilrite. The plantlets with well-developed shoot and root system were successfully established in garden soil and grown outside in a greenhouse with a 80% survival rate.     

In vitro clonal propagation of bird eye chilli (Capsicum frutescens Mill.)

An efficient and highly reproducible protocol for micropropagation of bird eye chilli Capsicum frutescens was attempted. Murashige and Skoog (MS) medium containing 0.5-3.0 mgl(-1) of 6-benzyladenine (BA), 2-isopentenyl adenine (2iP), kinetin and 0.5-2.0 mg l(-1) of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) along with 1 gl(-1) activated charcoal (AC) were used for shoot regeneration from both shoot tip and nodal explants. Shoot tip explants (100%) grew well on medium containing 1 mgl(-1) of kinetin and 1 mgl(-1) of IBA. Shoot proliferation (1-3) from nodal explants was effective on this medium. The regenerated shoots with 4-7 nodes had further growth upon sub-culturing onto kinetin (1 mgl(-1)) and IBA (1 mgl(-1)) and rooted simultaneously. The rooted plants were transferred to pots after hardening under controlled conditions. The survival percentage in pots was 80-90%.     

Micropropagation of Searsia dentata

A method for in vitro regeneration of Searsia dentata from nodal and shoot tip explants derived from mature trees is outlined. Nodal explants produced multiple shoots from the axis when cultured on Murashige and Skoog (MS) medium containing 3% sucrose supplemented with 0, 5, 7.5, 10, or 12.5 μM N ⁶-benzyladenine (BA). An average of 5.3 shoots was obtained from nodal explants on 10 μM BA. For shoot tip explants, however, supplementation of α-naphthaleneacetic acid (NAA) with BA favored a caulogenic response. A maximum of 6.1 shoots were produced per shoot tip explant on MS containing 7.5 μM BA plus 5.0 μM NAA. The in vitro-regenerated shoots produced roots when transferred to full-strength MS medium containing 3% sucrose and 10 μM indole-3-butyric acid (IBA). The developed plantlets were transferred initially to a mist house. After an initial acclimatization period of 3–4 mo, plantlets were shifted to the greenhouse where they thrived for 9 mo. The standardized protocol for mass propagation of S. dentata should eliminate the dependence on natural stands of plants for traditional medicinal purposes, and will also serve as a means of conservation as the species is heavily overexploited.     

Micropropagation of juvenile and adult Sorbus domestica L.

Successful propagation of seedlings and mature trees of Sorbus domestica L. has been achieved by in vitro methods. Multiple shoot formation was obtained by placing shoot apices or nodal segments on a modified Schenck and Hildebrandt medium containing benzyladenine. Regenerated shoots were excised and induced to root on media with auxin. In the best treatments 75–85% of shoots from juvenile material rooted. Rooting capacity of shoots from mature explants was lower (30%) and was not improved by dipping the base of shoots in concentration solutions of indolebutyric or naphthaleneacetic acids. Plantlets were ultimately established in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid     

An efficient in vitro regeneration protocol for Tagetes minuta

Tagetes minuta is a source of secondary products which are used as pharmaceuticals, pesticides and as flavour components in the food industry. Cotyledons and hypocotyls of T. minuta were cultured on MS medium with combinations of IAA or NAA and BA. Hypocotyl-derived callus developed adventitious shoots which failed to develop further. Cotyledon-derived callus, cultured on medium with IAA, regenerated adventitious shoots which developed into plantlets on MS medium or half-strength MS with 2.85 μM IAA. Cotyledons cultured on medium with 5.71 μM IAA + 44.4 μM BA and transferred to MS medium for shoot growth yielded the highest number of shoots. Nodal segments from developing shoots were micropropagated on half-strength MS medium with 2.58 μM IAA and 95% of plantlets produced adapted successfully to greenhouse conditions. In vitro plants micropropagated from nodes had many shoots whereas plants regenerated from shoot tips had only a single main stem. This difference in morphology was retained after two months growth in a greenhouse. There were no significant differences in leaf and shoot fresh and dry weights among the regenerated plants after two months growth. After six subcultures of cotyledon-derived callus on medium with IAA and BA all explants lost their ability to regenerate except those cultured on medium with 17.23 μM IAA and 44.4 μM BA. The methods of regeneration developed will facilitate selection of T. minuta plants more tolerant of environmental stress, their micropropagation, and the in vitro production of secondary products. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct Differentiation of Shoot Buds from Leaf Explants of Cajanus Cajan L.

A protocol was developed for direct differentiation of multiple shoot buds from leaf explants of Cajanus cajan. In a modified Murashige and Skoog's medium supplemented with 2.22 µM benzyladenine (BA), 0.57 µM indole-3-acetic acid (IAA) and 41 µM adenine sulphate (AdS), the segments of basal halves of the first two leaves of a young seedling incubated on filter paper bridges in liquid medium took 20 – 25 d to differentiate shoot buds. The explants after transfer to solidified medium, with lower concentration of BA (0.22 M) resulted in fast growing healthy shoots. The developed shoots (measuring ca. 3 cm) were rooted in a medium supplemented with 1.42 µM IAA. They were subsequently grown in pots with soil with more than 80 % transplantation success.     

Micropropagation of <Emphasis Type="Italic">Pueraria tuberosa</Emphasis> (Roxb. Ex Willd.) and determination of puerarin content in different tissues

Pueraria tuberosa, a medicinally important leguminous plant, yielding various isoflavanones including puerarin, is threatened, thus requiring conservation. In this study, fresh shoot sprouts of P. tuberosa, produced by tubers, were used as explants for in vitro micropropagation. Surface-sterilized nodal shoots were incubated on Murashige and Skoog (MS) medium supplemented with 8.88 μM benzyladenine (BA), 50 mg l⁻¹ ascorbic acid, and 25 mg l⁻¹ of each of citric acid and adenine sulphate. Cut ends of nodal stem segments rapidly turned brown, and cultures failed to establish. When 100 mg l⁻¹ ascorbic acid (ABA) and 25.0 mg l⁻¹ polyvinyl pyrrolidone (PVP) were added to the medium, explants remained healthy, and cultures were established. Bud-breaking of nodal stem explants resulted in multiple shoot formation. Shoots proliferated (35–40 shoots per culture vessel) on MS medium as described above, but supplemented with 4.44 μM BA and 0.57 μM indole acetic acid (IAA) and additives. After 4–5 passages, proliferating shoots exhibited tip-browning and decline in growth and multiplication. However, when shoots were transferred to fresh shoot proliferation medium supplemented with 2.32 μM kinetin (Kn), sustained growth and high rate of shoot proliferation (50–60 shoots per culture vessel) was observed. Shoots rooted when transferred to medium consisting of half- strength MS medium with 9.84 μM indole butyric acid (IBA) and 0.02% activated charcoal. Alternatively, individual shoots were pulsed with 984.0 μM IBA and transferred to glass bottles containing sterile and moistened soilrite. These shoots rooted ex-vitro and were acclimatized in the greenhouse. Plants were then analyzed for puerarin content using HPLC, and leaves showed maximum accumulation of purerarin.