<Emphasis Type="Italic">In vitro</Emphasis> bud regeneration of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis> and wild <Emphasis Type="Italic">Carthamus</Emphasis> species from leaf explants and axillary buds

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm⁻³ KIN or 0.5 mg dm⁻³ BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm⁻³ each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

Induction and differentiation of callus from embryos of Cocos nucifera L. by IAA-conjugates

Calli from young embryos of Cocos nucifera L. were induced on B5 medium supplemented with IAA-conjugates (IAA-asp or IAA-ala) at a concentration of 2.0 mg/1 and callusing was increased by about 10% if both IAA-conjugates, IAA-asp and IAA-ala were added together. Differentiation of shoots and roots was achieved by transferring calli to B5 medium supplemented with either IAA-asp (2.0 mg/1)+Kn(2.0 mg/1) or NAA (2.0 mg/1). Complete plantlets were obtained on B5 medium supplemented with NAA (0.5 mg/1)+BAP (2.0 mg/1)+PVP (1.0 g/1).Abbreviations IAA Indole-3-acetic acid - IAA-ala Indole acetyl-L-alanine - IAA-asp Indole acetyl-L-aspartic acid - Kn Kinetin - BAP N⁶-benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - NAA -naphthalene acetic acid - PVP Polyvinylpyrolidone     

Direct shoot regeneration from foliar explants of an epiphytic orchid,Acampe praemorsa (Roxb.) Blatter and McCann

An efficient and reproducible procedure is described for the large-scale propagation of an epiphytic orchid,Acampe praemorsa (Roxb.) B latter and McCann using foliar explants. Shoot buds were induced in basal parts of foliar explants on Murashige and Skoog medium supplemented with N⁶-benzyladenine (BA), kinetin (Kn) or thidiazuron (TDZ), the latter being most effective at 1.0 mg/1. Shoots formed to a TDZ-containing medium elongated following transfer to a substrate supplemented with 2.0 mg/l 1-naphthaleneacetic acid (NAA) and 0.5 mg/1 BA. NAA at lower concentrations had no beneficial effects on shoot regeneration, whether added to the medium along with BA, Kn or TDZ. However, it promoted shoot elongation and leaf expansion. Higher concentrations of NAA suppressed shoot regeneration. The frequency of shoot regeneration was greatly influenced by the developmental stage and orientation of the leaf. Shoots regenerated from the foliar explants were rooted successfully on MS medium containing 1.0 mg/l indole-3-butyric acid. The plantlets were acclimated and eventually transferred to a garden.Abbreviations BA N⁶-Benzyladenine - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) medium - NAA 1-Naphthaleneacetic acid - TDZ Thidiazuron (N-phenyl-N-1,2,3-thiadiazol-5-ylurea)     

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.     

Effect of plant growth regulators,temperature and sucrose on shoot proliferation from the stem disc of Chinese jiaotou (<Emphasis Type="Italic">Allium chinense</Emphasis>) and in vitro bulblet formation

In vitro shoot proliferation from stem disc of Allium chinense, a vegetatively propagated plant, was investigated in this experiment. In the present study, shoots were formed directly on stem discs on a medium containing 1 mg/l N⁶-benzyladenine (BA) and 0.5 mg/lα-naphthaleneacetic acid (NAA). These shoots were further cultured on MS media supplemented with various levels of BA in combination with NAA, and new shoot clusters developed easily from the explants cultured despite considerable differences in the induction of shoot clusters with different levels of BA and NAA. The most productive combination of growth regulators proved to be 1.0 mg/l BA and 1.0 mg/l NAA, in which about 17 shoots were produced per cluster in 8 weeks culture. Most of the formed shoots were rooted 15 days after being cultured on MS media supplemented with 0.1–1.0 mg/l NAA. The survival rate of the plantlets under ex vitro conditions was 95% in pots filled with a peat: sand (2:1 v/v) mixture after two weeks. In vitro bulblet formation were strongly promoted by the high temperature of 30°C compared to that at 25, 20 and 15°C, and 12% (w/v) sucrose appeared to be optimal for bulblet development. Results from this study demonstrated that A. chinense could be in vitro propagated by using stem discs and in vitro bulblet formation could be achieved.     

Influence of some plant growth regulators on the growth and organogenesis ofCymbidium aloifolium (L.) Sw. seed-derived rhizomesin vitro

Summary An efficient procedure is outlined forin vitro regeneration of an epiphytic orchid,Cymbidium aloifolium (L.) Sw. using rhizomes developed from seeds. Murashige and Skoog's (1962) medium (MS) containing indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or 1-naphthaleneacetic acid (NAA) stimulated growth and proliferation of rhizomes with NAA being most effective at 5.0 mg.l⁻¹ (27.0 μM). Shoot bud differentiation was induced in the apical portions of the rhizomes on MS medium containing kinetin (Kn) or N⁶-benzyladenine (BA). The highest frequency of shoot regeneration (91.5%) and the maximum number of shoot buds formed (3.5 shoots/rhizome) were recorded with BA at 1.0 mg.l⁻¹ (4.4 μM). NAA (0.1 mg.l⁻¹, 0.54 μM), whenever added to the medium in conjunction with BA (1.0 mg.l⁻¹, 4.4 μM), slightly enhanced the frequency of shoot bud regeneration (92.6%) and the number of shoot buds formed (5.2 shoots/rhizome). Moreover, an NAA-BA combination induced rooting in regenerated shoots thereby producing complete plantlets in one step. Shoots developed on cytokinin-supplemented medium were rooted on MS containing NAA at 1.0 mg.l⁻¹ (5.4 μM). Regenerated plantlets were acclimated and eventually established in a garden.     

High frequency shoot regeneration from nodal and shoot tip explants in Holarrhena antidysenterica L

Shoot tip and nodal segment explants of Holarrhena antidysenterica when cultured on MS medium containing BAP (1.0-3.0 mg/l) with NAA (0.2-1.0 mg/l) and BAP (1.0-3.0 mg/l) with Kn. (0.2-1.0 mg/l) produced multiple shoots. Maximum multiple shoots was found in MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l). Subculture on the same medium resulted in rapid shoot multiplication at an average rate of 16 new shoots per subculture. Addition of urea (100 mg/l) in the medium increased the number of shoots up to 22 per culture. For best rooting, the shoots were excised from the culture flask and implanted individually on half strength MS medium with 0.5 mg/l each of IBA, IAA and NAA. After 20 days of transfer on root induction medium 95% rooting was achieved. Regenerated plantlets were successfully acclimatized and established in soil. About 90% of plantlets survived under open field conditions.     

Micropropagation of Sesbania sesban from the Mature Tree-Derived Explants

The nodal and internodal explants excised from the orthotropic shoots of Sesbania sesban var. bicolor elicited the development of shoots directly from the explants as well as via an intervening callus phase on Nitsch (N) medium. On benzyladenine (BA) supplemented media, the adventitious shoot buds developed involving a callus phase. An average of 8.9 ± 4.1 shoots developed per nodal explant on N medium containing 0.5 mg dm⁻³ BA in 95 % cultures, whereas 65 % cultures of internodal explants developed shoots with an average of 5.9 ± 3.6 shoots per explant on N medium supplemented with 1.0 mg dm⁻³ BA. On kinetin (Kn) supplemented medium shoots developed directly from the surface of both the explants at all the concentrations tried. Nodal explants on N medium supplemented with 1.5 mg dm⁻³ Kn developed an average of 12.5 ± 7.9 shoots per explant in 100 % cultures, while internodal explants induced an average of 11.6 ± 7.4 shoots per explant in 75 % explants at 0.5 mg dm⁻³ Kn. The in vitro regenerated shoots developed roots when implanted on N medium supplemented with 2 mg dm⁻³ indole-3-butyric acid (IBA), after 30 d of inoculation. The in vitro developed plantlets were initially acclimatized under controlled conditions for four months, prior to their transfer to the field. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic embryogenesis and in vitro flowering of 3 species of bamboo

Plant regeneration via somatic embryogenesis was achieved in callus cultures derived from nodal explants of in vitro grown seedlings and excised mature zygotic embryos of three bamboo species on Murashige and Skoog's (MS) basal medium supplemented with 0.5 mg/l kinetin (Kn), 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 10 mg/l adenine sulphate (Ads) and 3% (w/v) sucrose incubated in the light or in the dark. Somatic embryos germinated (95–98%) into normal plants and were transferred to soil with 95% success. In vitro flowering was induced on shoots developed from nodal explants taken from somatic embryo regenerated plants of Bambusa vulgaris, Dendrocalamus giganteus and Dendrocalamus strictus on half-strength MS basal medium supplemented with 0.25 mg/l indole-3-butyric acid (IBA), 0.5 mg/l Ads, 0.5 mg/l gibberellic acid (GA₃) and 3% sucrose.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - Ads adenine sulphate - IBA indole-3-butyric acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) basal medium - GA₃ gibberellic acid     

Rapid clonal propagation of Saccharum officinarum L. Vars. CO-6907 and CO-86249 and to assess the genetic uniformity through molecular markers

Abstract

An efficient protocol was developed for in vitro clonal propagation of Saccharum officinarum Vars. CO-6907 and CO-86249 through axillary meristem culture. Maximum meristem elongation was achieved on Murashige and Skoog's (MS) medium supplemented with 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kn) within 15 days of culture. Multiple shoots were induced from meristems on MS basal medium supplemented with 1.0 mg/L BA, 0.5 mg/L Kn, 0.25 mg/L 1-napthaleneacetic acid (NAA) and 3% (w/v) sucrose. Addition of 0.1–0.25 mg/L gibberellic acid into the multiplication medium found the better shoot elongation. Repeated subculture on multiplication medium induces higher rate of shoot multiplication. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 1.0–2.0 mg/L NAA or indole-3-butyric acid and 6% (w/v) sucrose. While either decreasing or increasing of sucrose concentration in the rooting medium, the percentage of rooting was reduced. Maximum percentage of rooting was achieved on medium having 2.0 mg/L NAA with 6% (w/v) sucrose. About 80% of micropropagated plantlets were hardened in the greenhouse and successfully established in the soil. Random Amplified Polymorphic DNA marker was used to detect the variability among the micropropagated plants developed through in vitro. The results showed that there was no polymorphism among the micropropagated plants. This study will help for propagation of quality planting material of high-yielding variety of sugarcane for commercialization.     

Differential cytokinin effects on the stimulation of in vitro shoot proliferation from meristematic explants of castor (Ricinus communis L.)

A highly efficient and reproducible method of in vitro propagation using meristematic explants has been developed for castor. Embryo axes and shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 0.5–10.0 mg/l of adenine, N⁶-benzyladenine (BA), kinetin (Kn), thiadiazuron (TDZ) and zeatin. TDZ (1.0–10.0 mg/l) gave the maximum number of shoots (37.8–40.0) from embryo axes, while BA (2.0 mg/l) was found superior to other cytokinins for obtaining the highest number of shoots (46.7) from the shoot apex. Adenine and Kn at all of the tested concentrations resulted in low proliferation rates from embryo axes. The carryover effect of the cytokinins was tested by subculturing proliferating shoot cultures from various media onto the medium fortified with 0.5 mg/l BA. There was no significant influence of the cytokinins on subsequent proliferation from the two explant types except for TDZ with embryo axes. The number of shoots from TDZ-habituated embryo axes ranged between 36.0 and 81.7, while it varied from 5.7 to 22.0 and 3.7 to 28.3 in axillary buds and embryo axes, respectively, on the other media. For elongation of shoots, gibberellic acid (GA₃) (0.1–1.0 mg/l) was added to the medium supplemented with 0.2–0.5 mg/l BA. Incorporation of GA₃ (0.1 mg/l) significantly enhanced the frequency of elongated shoots but drastically reduced the multiplication ability. Hence, proliferating shoot clusters were periodically transferred to the medium supplemented with 0.5 and 0.2 mg/l BA for further multiplication and elongation. Well-developed shoots were rooted on half-strength MS medium supplemented with 1.0 mg/l indole-3-butyric acid. The rooted plantlets were acclimatized with more than 60% success. Received: 17 June 1997 / Revision received: 3 September 1997 / Accepted: 20 September 1997     

In vitro culture of Cuminum cyminum regeneration of flowering shoots from calli of hypocotyl and leaf explants

Calli from hypocotyl explant of Cuminum cyminum L. (Cumin) grew rapidly on Gamborg's B₅ basal medium with the following supplements, (i) 0.5 mg/l — 2,4-D (ii) 4 mg/l — NAA plus 2 mg/l — Kinetin and (iii) 0.2 mg/l — NAA plus 0.2 mg/l — BAP, whereas calli from leaf explant in these media grew slowly. Hypocotyl and leaf calli produced roots when transferred to basal medium only and shoots in basal medium with 0.5 mg/l NAA and 0.1 mg/l BAP. Ninety percent of the shoots produced roots when they were transferred to half strength MS inorganic salts supplemented with 0.5 mg/l each of IBA and NAA.Fifty to sixty percent of rootless as well as rooted shoots produced terminal umbellate flowers on this medium.     

Organogenesis and tuberization in cultures of Dioscorea floribunda

Callus cultures were established from node and internode segments of Dioscorea floribunda Mart. & Gal. Both Murashige and Skoog's and modified White's medium supported callusing as well as organogenesis when supplemented with either 2,4-D or NAA in combination with BAP or Kn. On development of shoot primordia, calli were transferred to unsupplemented, half strength MS basal medium. This procedure led to the increase in formation of shoots. Several crops of shoots were obtained from single differentiating callus cultures by excising the shoots and subculturing the residual part. Seventy percent of plantlets survived rooting and transfer to soil.When they were maintained in half-strength MS basal medium and 0.5 mg1^-1 of NAA, 70% of plantlets formed aerial tubers at nodes. These tubers produced both roots and shoots and could be detached from the mother plant.     

Micropropagation of globe artichoke (Cynara scolymus L.) from underground dormant buds (“ovoli”)

Summary Side shoots excised from underground dormant buds ofCynara scolymus L. were used as primary explants to establishin vitro cultures. A 3×3 factorial experiment with all possible combinations of three concentrations (0.5, 1.0, 2.0 mg/liter or 2.22, 4.44, 8.88 μM) ofN ⁶-benzyladenine (BA) and three concentrations (0, 0.1, 0.2 mg/liter or 0, 0.54, 1.07 μM) of 1-naphthaleneacetic acid (NAA) was used to determine the optimum growth regulator combination for shoot multiplication. The highest rate of axillary shoots was induced on Murashige and Skoog agar medium supplemented with 0 mg NAA/liter and 1.0 mg BA/liter (4.44 μM). Other cytokinins tested (kinetin, zeatin, and 2-isopentenyl-adenine were less effective than BA in inducing axillary shoot growth. Up to 60% of elongated microshoots rooted after 5 weeks on 1/2 MS agar medium supplemented with 2 mg/liter (11.42 μM) indole-3-acetic acid (IAA). Seventy percent of rooted plantlets were transferred successfully into soil. Plants are under evaluation for their genetic uniformity and clonal fidelity.     

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.     

Plant Regeneration Through Somatic Embryogenesis in Leaf Derived Callus of Plumbago Rosea

Regeneration of Plumbago rosea L., a rare medicinal plant, via somatic embryogenesis in callus cultures derived from leaf explants was described. Optimum callus formation was achieved on semi-solid Murashige and Skoog (MS) medium supplemented with 0.25 mg dm^–3 kinetin and 2.0 mg dm^–3 1-naphthaleneacetic acid (NAA). Somatic embryogenesis was achieved upon transferring the 4-week-old callus to a medium containing 1.0 mg dm^–3 kinetic (Kn), 0.5 mg dm^–3 gibberellic acid (GA₃) and 0.1 mg dm^–3 NAA. Embryo maturation and germination was achieved on the half-strength MS basal salts supplemented with 0.01 – 0.25 mg dm^–3 Kn and 2 % (m/v) saccharose. An average of 50 – 60 plantlets were obtained from 150 mg of embryogenic callus within 4 week of subculture. Out of the 50 plantlets about 28 survived in the greenhouse.     

In vitro Clonal Propagation and Organogenesis in Spilanthes acmella (L) Murray: A Herbal Pesticidal Plant of North-East India

Multiple shoots were regenerated in MS medium using different concentrations of BAP and Kn and different combinations of BAP with IAA, NAA and IBA. Highest multiplication of shoots was obtained with BAP (0.75 mg l^?1) with 28.4 shoots per explant after 60 days of culture. Shoots rooted best on IBA (0.5 mg l^?1), numbering 48.8 per explant. Organogenesis was maximum in callus cultured on MS medium supplemented with BAP (2.0 mg l^?1) and IAA (1.0 mg l^?1).     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

Induction of root and shoot formation from root meristems ofPetunia hybrida

Summary A procedure has been developed for the induction of root or shoot formation from root meristems of germinated seeds ofPetunia hybrida. Root formation was obtained on Murashige and Skoog (MS) medium supplemented with a combination of 6-benzylaminopurine (BA) (0–0.5 mg/l) and naphtaleneacetic acid (NAA) (0.05–2.0 mg/l). Induction of predominantly shoot formation was obtained on MS medium containing the following combinations of hormones (in mg/l): 0.05–0.5 NAA and 0.25–2.0 BA. Complete plant formation was obtained after rooting of the shoots on MS medium supplemented with IAA (0–2.0 mg/l) or NAA (0-0.5 mg/l).