In vitro flowering and production of viable pollen of cucumber

Flowers were produced on sterile cucumber (Cucumis sativus L.) plants grown in vitro from seed and micropropagated shoots from stem fragments. The highest numbers of flowers on plants from both sources were produced on Murashige and Skoog (MS) medium without plant growth regulators (PGR), as well as with 6 μM of kinetin (Kin). Plants cultured on MS medium supplemented with 8.9 μM benzyladenine (BA) and 1.1 μM 1-naphthaleneacetic acid (NAA) did not flower. In vitro grown plants produced fewer, smaller flowers compared with greenhouse-grown plants. Male and female flowers developed on plants grown in vitro from seed and were morphologically similar to flowers on greenhouse grown plants. Micropropagated shoots produced male flowers with altered morphology. The highest viability (72.9 ± 4.2%) and germination (69.5 ± 4.1%) of pollen were observed for plants grown from seed on MS medium supplemented with 6 μM Kin. Cytological observations of meiosis in anthers of male flowers from in vitro grown plants revealed abnormalities, such as monads, dyads, triads, polyads, microcytes and degeneration of tetrads, causing reduced viability and germination of pollen. The fewest meiotic irregularities in pollen mother cells were observed in plants grown on MS medium that was PGR-free (12.1 ± 0.9%) or with 6 μM Kin (20.9 ± 1.7%).     

Plantlet Regeneration via Multiple Shoot Induction in Indian Cultivars of Lucerne (Medicago sativa L)

An efficient protocol has been developed for in vitro plant regeneration via multiple shoot induction in lucerne (Medicago sativa L). Shoot tips from in vitro grown 5–6 days old seedlings of 3 cultivars, LLC-3, Chetak and RL-88 were used as explants for multiple shoot induction on MS medium supplemented with cytokinins. Maximum of 14 shoots per apical meristem were observed in case of cv Chetak on MS medium supplemented with BAP (12.6 μM) and KN (9.3 μM). Shoot elongation on MS medium supplemented with GA (5.8 μM), while root induction was achieved on MS medium supplemented with IAA (11.4 μM) and activated charcoal (2.0 g l^?1). Tissue raised plants showed 75% survival after transfer to soil under field conditions.     

In Vitro Micropropagation and Flowering in Artemisia annua

A protocol for quick regeneration of a large number of plantlets of Artemisia annua (source of a potent antimalarial drug) is being reported. Multiple shoots were obtained in large numbers from juvenile as well as vegetative parts of mature plant on Murashige and Skoog’s medium (MS) having 3% sucrose and 800 μM myoinositol and supplemented with NAA (0.5 μM) + BAP (13.0 μM) + GA₃ (0.3 μM) + Asp (300.0 μM) + Glu (700.0 μM) + Arg (300.0 μM) + Cys - HCl (30.0 μM). Reversal of reproductive to vegetative phase and back to reproductive phase could be achieved in the cultured flower buds. The shoots obtained on the above medium could be induced to flower. In addition, new shoots that differentiated from vegetative parts of juvenile and mature explants also produced flowers when cultured on MS with GA₃ (0.3 μM). Since artemisinin estimation is correlative to flowering, our results would facilitate better understanding of biosynthesis of this drug in vitro.     

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.     

In vitro propagation of Caralluma tuberculata and evaluation of antioxidant potential

Present study describes rapid in vitro propagation of Caralluma tuberculata, a traditional medicinal plant, and antioxidant potential of calli and plants extracts. The highest callus induction rate (93.3%) with maximum weight of calli 5.2 g was achieved from shoot tip explants on MS medium supplemented with 9.04 μM 2,4-D and 4.44 μM BA. The maximum shoot induction rate (71.1%) with mean number of shoots 3.66 ± 1.53 and 4.6 cm average shoot length was observed on 13.32 μM BA, 4.52 μM 2,4-D and 2.89 μM GA3 appended in MS medium. The developed shoots were best rooted in the presence of 5.07 μM IAA with 3.0 ± 0.15 roots per plantlet. The plants were successfully acclimatized under in vivo conditions. The plants and calli extracts exhibited good antioxidant activities, however, plant extract activities were more pronounced. The phenolic compounds in plant and calli extracts were 0.16% and 0.057%, respectively. While the flavonoids were 0.092% in plant and 0.039% in calli extract. Total Phenolics, flavonoids; DPPH radical scavenging activity and reducing power potential distributed among different fractions depending upon polarity of the solvent. The highest DPPH scavenging activity and reducing power was exhibited by water fractions; 4.95 mg/mL and 0.729 OD at 10 mg/mL, respectively. The micropropagation protocol can be successfully used for large-scale multiplication and conservation of germplasm of this threatened plant. Furthermore, antioxidant value describes importance of this valuable plant as food and medicine.     

Efficient in vitro propagation of <Emphasis Type="Italic">Artemisia nilagirica</Emphasis> var. <Emphasis Type="Italic">nilagirica</Emphasis> (Indian wormwood) and assessment of genetic fidelity of micropropagated plants

A reliable protocol has been established for in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood), a valuable medicinal plant from India. A highly proliferating organogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 2.5 µM IAA when nodal explants were cultured on MS medium supplemented with various growth regulators. Further, highest regeneration frequency (83.3 %) of adventitious shoots was observed, when the callus was sub-cultured on MS medium supplemented with 6-benzylaminopurine (BAP; 2.5 µM) along with 7.5 µM 2-isopentenyl adenine (2-iP). An optimal of 10.16 ± 2.24 shoots were regenerated on medium supplemented with 2.5 µM BAP + 7.5 µM 2-iP. Quarter strength MS medium supplemented with 10 µM IBA was effective for rooting of the shoots. Ex-vitro plants were normal and were established successfully. Cytological and molecular marker studies showed that regenerated plants showed genetic stability in micro-propagated plants.     

Influence of plant growth regulators on micropropagation and in vitro flowering of Trichodesma indicum (Linn) R. Br

An efficient protocol for micropropagation and in vitro flowering of Trichodesma indicum (Linn) R. Br. was developed using shoot tip explants. The physiological role of cytokinin and its combination with auxins on micropropagation and in vitro flowering was investigated. The highest number of shoots (9.94 ± 0.10) and the maximum average shoot length (5.56 ± 0.35 cm) were recorded on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP) (4.44 μM) and naphthaleneacetic acid (NAA) (2.69 μM). The effect of sucrose concentration on in vitro floral development was studied in plantlets cultured on MS medium supplemented with gibberellic acid (GA₃) and BAP. The highest percentage of flowering (93.2%) was obtained on MS medium supplemented with GA₃ (1.44 μM), BAP (1.33 μM) and sucrose (30 g l^?1). Root formation from the adventitious shoots was easily achieved on MS medium containing indole-3-butyric acid (IBA) (2.46 μM). The regenerated plantlets showed 86% survival rate and were phenotypically normal. The described method can be successfully employed for large-scale multiplication and in vitro flowering of T. indicum.     

Embryogenesis and plant regeneration from unpollinated ovary culture of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis>

Embryogenesis and plant regeneration was achieved from callus cultures derived from unpollinated ovaries of Psoralea corylifolia L. Callus was initiated from unpollinated ovaries on Murashige and Skoog (MS) medium supplemented with 2.2 μM N ⁶-benzyladenine (BA) and various concentrations of α-naphthaleneacetic acid (NAA (2.7 to 10.7 μM) or 2,4-dichlorophenoxyacetic acid (2,4-D (2.3 to 9 μM) alone or in combination. Highly organized embryogenic callus induction, embryo development, proliferation and maturation were achieved on transfer of callus clumps to MS medium supplemented with NAA (0.27 μM) or 2,4-D (0.23 μM) alone or in combination with BA (2.2 to 8.8 μM). Addition of abscisic acid (ABA) (0.95 to 5.8 μM) to the medium enhanced average numbers of cotyledonary stage embryos, the maximum number (34.6 ± 0.7) being obtained on MS medium containing 0.27 μM NAA, 2.2 μM BA and 3.8 μM ABA. Embryos germinated on MS medium supplemented with BA (0 to 8.8 μM). MS medium containing gibberellic acid (GA₃ (0.29 to 5.8 μM) enhanced embryo germination frequency, the highest frequency (66.7 %) occurring on MS medium containing 2.2 μM BA and 4.3 μM GA₃. Effect of several concentrations (3.0 to 6.0 %) of sucrose or maltose was also observed on germination of embryos. MS medium enriched with maltose supported high frequency of embryo germination.     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.     

Rapid In Vitro Propagation of Eclipta alba (L) Hassk by Shoot Tip Culture

An efficient and rapid tissue culture system employing shoot tip explants has been developed for Eclipta alba (L) Hassk, an important medicinal plant of the family Asteraceae. The highest shoot regeneration frequency (95%) as well as the maximum number (32.2 ± 0.4) of shoots was recorded on MS medium amended with BA (5 μM) and NAA (0.5 μM). The regenerated shoots rooted best on MS medium supplemented with 0.2 μM IBA. The in vitro developed plantlets were acclimatized successfully with 100 % survival.     

Plant regeneration via somatic embryogenesis from solid and suspension cultures of Vitis vinifera L. cv. ‘Manicure Finger’

Vitis vinifera L. cv. ‘Manicure Finger’ is one of the major table grape varieties in China. To provide a strong foundation for genetic transformation with potential for crop improvement, we undertook plant regeneration via somatic embryogenesis. Anthers and gynoecia were harvested from immature flowers and used as explants to induce embryogenic calli. Explants cultured in MS1 medium (based on Murashige and Skoog basal salts), supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4-μM 6-benzylaminopurine (6-BA) showed the highest rates of embryogenic callus induction (3.7%?±?1.3% for anthers and 4.8%?±?2.5% for gynoecia). After several months, somatic embryos were produced from embryogenic calli cultured in plant growth regulator-free MS2 medium (with reduced sucrose). Somatic embryos (SE) at the cotyledonary stage were isolated and cultured on three different media (MS2, MS3, or B) for conversion into plantlets, the efficiency of which ranged from 63.9%?±?4.8% to 83.9%?±?8.4%. After 1 mo of in vitro culture, 80% of plants with at least six leaves were successfully transplanted into soil. SE was repeatedly induced from previously induced somatic embryos for up to 1.5 yr. Using embryogenic calli as starting material, suspension cultures containing embryogenic cell aggregates were also established in liquid MS medium supplemented with 4.5-μM 2,4-D. The embryogenic cell aggregates continued to proliferate without differentiating for successive subculture cycles. After transfer to 2,4-D-free liquid medium for 4 wk, an average of 63.7%?±?9.0% mature SEs were produced per 20 mL of liquid medium. More than 40% of somatic embryos at cotyledonary stage, derived from the suspension cultures, successfully germinated into plants using solid medium.     

Improved plant regeneration in Capsicum annuum L. from nodal segments

Multiple shoots were induced by culturing nodal explants excised from 1-month-old aseptic seedlings of red pepper (Capsicum annuum L. cv. Pusa Jwala) on Murashige and Skoog (MS) medium supplemented with (0.1–10 μM) thidiazuron (TDZ). The rate of multiple shoot induction per explant was maximum (14.4 ± 0.06) on MS medium supplemented with 1.0 μM TDZ. Regenerated shoots were elongated well on growth regulator free MS medium. Adventitious roots were induced two weeks after transfer of elongated shoots to MS medium supplemented with auxins (IAA, IBA or NAA) in different concentrations. Optimum root formation frequency was obtained in medium containing 1.0 μM IBA. Ex-vitro rooting was also achieved by pulse treatment with 300 μM IBA for 10 min. Rooted shoots were transplanted in plastic pots containing garden soil (with 90 % survival rate), where they grew well and attained maturity. Regenerated plants were phenotypically and cytologically normal.     

Regeneration of Indian ginseng plantlets from stem callus

The development of stem callus mediated plant regeneration system for Withania somnifera is described. Maximum callus proliferation was obtained on Murashige and Skoog medium supplemented with 2.26 μM 2,4-D. Three-week-old, white, friable callus was used for shoot regeneration. The maximum shoot regeneration (6.2 ± 0.34 shoots/explant) was achieved in four weeks when callus was cultured on MS medium fortified with 4.44 μM BA and 0.57 μM IAA. Regenerated shoots were excised and multiplied (8.4 ± 0.43 shoots/explant) on MS medium supplemented with 4.44 μM of BA. Multiple shoots were divided into single shoots and were rooted (5.1 ± 0.49 rootlets/shoot) on half strength MS medium supplemented with 9.84 μM of IBA. After a hardening phase of 3 weeks the plantlets were transferred to the field. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro cormlet production of saffron (Crocus sativus L. Kashmirianus) and their flowering response under greenhouse

A complete protocol for the saffron cormlet production under in vitro conditions and subsequent flowering under greenhouse conditions is described. Highest number of cormlets (70.0 ± 0.30) per corm slice (explant) could be regenerated on Murashige and Skoog (MS) half strength medium supplemented with thidiazuron (TDZ) (20 μM), Indole acetic acid (IAA) (10 μM), and sucrose (40 g/l). Maximum germination (90%) of these cormlets could be achieved on MS medium containing 6-benzyl amino purine (BAP) (20 μM) and α-naphthalene acetic acid (NAA) (15 μM). In order to increase the size of the in vitro raised cormlets, these were cultured on MS medium containing TDZ (15 μM) and IAA in the range of 1.5-30 μM. Maximum increase in cormlet size could be attained on TDZ (15 μM) + IAA (12.5 μM) + sucrose (30 g/l), and the average size of cormlets was 2.5g. In another experiment, apical vegetative buds of actively growing corms were cultured for cormlet development, and corms of size 2.5g could be developed on MS medium with NAA (15 μM), BAP (20 μM), and sucrose (30 g/l). The in vitro developed cormlets were dried under shade at 25 ± 2°C for 7 d. These were then planted in small cups containing clay loam soil and kept in green house at 20 ± 2°C. In vitro developed cormlets with mean weight 2.5 g showed maximum flowering (25%) as well as vegetative growth (55%), while only 19% cormlets of 2.0 g flowered. To our knowledge this is the first report on successful flowering from in vitro raised cormlets under greenhouse.     

High-frequency<Emphasis Type="Italic">in vitro</Emphasis> flowering in six species of<Emphasis Type="Italic">ceropegia</Emphasis>

High-frequencyin vitro flowering is reported here fromin vitro regenerated shoots ofin vitro-raised seedlings of rare and endemicCeropegia lawii, Ceropegia maccannii, Ceropegia oculata, andCeropegia sahyadrica, as well as the widely distributedCeropegia bulbosa var.bulbosa andCeropegia hirsuta. In our first set of experiments, the MS medium contained 87 mM sucrose and was supplemented with varying concentrations of BAP (4.4 to 26.6 μM). For the second set of trials, varying concentrations of sucrose (87 to 233 mM) were tested in MS media containing a constant 4.4 p.M BAP. Sub-cultured apical as well as axillary buds flowered with similar frequencies after 30 d of incubation. For all six species, the highest percentage of flowering shoots was obtained with either 26.6 μM BAP or 175 mM sucrose. Although smaller in size, theirin vitro flowers were morphologically comparable within wVo-derived flowers. Variations among species were noted for the number of flower buds per shoot and the percentage of flower formation. Because all six species showed similar responses in both experiments, we can suggest that this protocol is applicable across the wide range ofCeropegia species.     

麝香石竹花芽离体发育的阶段控制

在离体条件下,利用不同的培养基对麝香石竹顶芽外植体的花芽发育进行了阶段控制的研究.结果表明,麝香石竹的顶芽外植体在MS+KT1.0mg/L+IAA1.0mg/L+蔗糖3.0%+琼脂0.8%的Ⅰ级培养基上能被诱导花芽发育的启动;然后,将已诱导花芽发育启动的顶芽外植体,转接到MS+KT1.0mg/L+IAA0.5mg/L+蔗糖1.5%+葡萄糖1.5%+琼脂0.8%的Ⅱ级培养基上能进行花芽的进一步发育形成花蕾,且能从一个花蕾继续分化发育重新产生2—3个花蕾;把花蕾再转接到改良的MS+BA2.0mg/L+NAA0.2mg/L+蔗糖1.5%+葡萄糖1.5%+琼脂0.8%的Ⅲ级培养基上,培养一周后花蕾的花瓣张开,花朵全部开放.不同麝香石竹品种,诱导花芽发育启动的效果不同,Scania品种诱导效果最好.花芽发育初期可溶性蛋白含量较高,但随着花芽发育的进程而迅速下降,不同花芽发育时期的过氧化物酶活性均强于营养器官.本文为花芽分化发育机理的研究创造了条件,也为鲜花生产探索了新路子.     

Micropropagation and in vitro flowering of endemic and endangered plant Ceropegia attenuata Hook

Factors affecting in vitro propagation were evaluated for Ceropegia attenuata Hook., an endemic and endangered plant having ornamental potential but a limited reproductive capacity. Rapid shoot multiplication from nodal explants was established using varying concentrations of cytokinins and auxins either alone or in combinations. The highest frequency of shoot induction was achieved when nodal explants were inoculated on Murashige and Skoog (MS) medium supplemented with 13.31 μM 6-benzylaminopurine with a mean of 12.9?±?0.5 shoots per explant. High concentrations of TDZ (6.81–11.35 μM) and KN (6.78–11.61 μM) resulted in stunted and vitrified shoots. Factors implicated in the promotion of floral transition of the C. attenuata have been identified which are 4-amino-3, 5, 6-trichloropicolinic acid (picloram), 6-benzylaminopurine, sucrose and photoperiod. The highest frequency of flowering (100%) was obtained when axillary shoot explants were transferred to MS medium supplemented with picloram (4.14 μM) within 4 weeks of culture. Transfer of in vitro regenerated shoots to half strength MS medium with 2.46 μM indole-3-butyric acid (IBA) showed maximum root induction. The in vitro grown plantlets were successfully acclimatized in the glasshouse with 85% of survival and showed normal development. The developed protocol provided a simple, cost-effective approach for the conservation of endangered plant C. attenuata for replenishing its declining populations.     

Hormonal Control of Sex Differentiation of Potentially Female Floral Buds of Lagenaria siceraria var. hispida In Vitro

In the present study, several kinds of phytohormones were used for the control of sex differentiation of the potentially female floral buds of Lagenaria siceraria var. hispida in vitro. It was shown that both GA3 and STS (silver thiosulphate) could effectively change the direction of sex differentiation of the potentially female floral buds in vitro. In the MS medium, supplemented with IAA, BA and ACC(1-aminocyclopropane-l-carboxylic acid) at l nmol/L, male flowers would be induced from the potentially female floral buds by the addition of GA3 (1–500μmol/L). Herein the male flowers were induced more effectively by GA3 within 5–-20 μmol/L but it was not as effective as STS. In the MS medium supplemented with IAA, BA and ACC at 1 nmol/L and with GA3 at 20 nmol/L more male flowers were differentiated from the potentially female floral buds with the addition of STS within 100–500 μmol/L. On the contrary, when the MS medium were supplemented with IAA and ACC at 1 nmol/L and with BA increased to 100 nmol/L more female flowers were differentiated from the potentially female floral buds, even with addition of 10–50 nmol/L of GA3.     

Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers

This study describes an efficient plant regeneration protocol for Abutilon indicum via somatic embryogenesis from 2,4-dichlorophenoxyacetic acid (2,4-D)-induced leaf-derived callus on MS medium, fortified with 13.32 μM 6-benzyladenine (BA), 2.68 μM α-naphthalene acetic acid (NAA), 200 mgl^?1-activated charcoal, and 11.54 μM ascorbic acid. This combination produced the highest (15.5 ± 0.7) number of somatic embryos after four weeks of culture. Further, the embryogenic calli were transferred to MS medium supplemented with 13.32 μM BA, 1.44 μM gibberellic acid (GA₃), and 3% (w/v) sucrose and showed highest rate of germination (76.3 ± 7.0%). The germinated somatic embryos showed maximum plantlet conversion (62.6 ± 1.90%) on ½ MS medium supplemented with 4.92 μM indole-3-butyric acid and 6.0% sucrose (w/v). The highest frequency of secondary somatic embryogenesis (34.4 ± 0.82) was observed on ½ MS medium, supplemented with 133 μM FeSO₄·7H₂O, 74 μM ethylene diamine tetraacetic acid disodium dihydrate (disodium EDTA), and 15% polyethylene glycol-4000 (PEG-4000) after three weeks of subculture. Scanning electron microscopy observations also substantiated the development of primary and secondary somatic embryos from embryogenic calli. Start codon targeted polymorphism (SCoT) marker analysis of 214 somatic embryo-derived plantlets amplified 167 numbers of bands ranging from 230 to 2125 bp. The homogeneous banding pattern confirmed the genetic uniformity of this sample of somatic embryo-derived plantlets as compared with the donor plant.     

Silver nitrate-induced in vitro shoot multiplication and precocious flowering in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don,a rich source of terpenoid indole alkaloids

Catharanthus roseus (L.) G. Don is an economically and medicinally important plant since its leaves and flowers contain terpenoid indole alkaloids. The present study, for the first time, encompasses the influence of silver nitrate (AgNO₃), in consort with cytokinins like N ⁶-benzyladenine (BA) and 6-furfurylaminopurine (kinetin), to regenerate multiple shoots from nodal segments explants and to induce high-frequency precocious flowering of C. roseus under in vitro condition. Synergistic effect of equal concentrations of BA and kinetin was enhanced following the amalgamation of AgNO₃. As high as 98% explants responded to multiple shoot initiation and proliferation in Murashige and Skoog medium supplemented with 3 µM BA, 3 µM kinetin and 0.1 µM AgNO₃. As many as 7 shoots were developed per explant following 12 days of inoculation. Continuous culture in the same medium for 21 days induced precocious flowering from 75% shoots, wherein a maximum of ~?6 (5.67?±?0.88) flowers was observed per in vitro shoot. On the other hand, in the combinations of BA and kinetin excluding AgNO₃, a maximum of 6.67% explants responded and initiated merely 3.33 shoots per explant. Nevertheless, no induction of flower was observed in the media devoid of AgNO₃. Our results on the induction and proliferation of multiple shoots with simultaneous flowering would help the global pharmaceutical industry to produce in vitro shoots and flowers in bulk, as an alternative source of alkaloids.