Shoot Tip Explants Research Articles

Effect of auxin on in vitro rooting of the triploid Musa paradisiaca L. cv. Nendran (AAB)

Banana is an important fruit crops of the family Musaceae. Tissue culture multiplication of banana is now popular and preferred due to faster multiplication, uniformly and cost-effective production of healthy planting materials. The following work was take on to study the result of various concentrations of auxin (indole-3-butyric acid-IBA). Shoots derived from sucker explants after a series of multiplication (4 to 5), regenerated plants having 4.5 cm length were dissected out and rooted in different concentrations of auxin (IBA) (0.5 to 3.5 mg/L). Root production was started after 10 days of culture and the data taken after 20 days. From all the treatment tested all media concentrations produce roots at varied level with in a period of 15-20 days of culture. Media having concentrations of IBA were used to induce in vitro rooting in plantlets of Musa cv. Nendran after six sub cultures including elongation. The elongated shoots harvested from culture bottles were transferred to MS solid media supplemented with auxin IBA at varied concentrations. The root induction rate varied from one media concentration to another. The present study revealed that IBA (1.0 mg/L) was found to be most applicable hormone for root induction in sucker shoot tip explants of Musa cv. Nendran.

Phytochemical characterization of callus cultures from the endangered plant Crocus scepusiensis (Rehm. & Woł.) Borbás ex Kulcz.

Crocus scepusiensis (Rehm. & Woł.) Borbás ex Kulcz., a critically endangered herbaceous plant which serves as a valuable source of bioactive compounds found across Europe and Asia. The aim of this study was to produce a calli from two different plant parts (leaf and shoot tip) for the critically endangered C. scepusiensis through tissue culture techniques, characterize the resulting calli through chemical profiling, with a focus on identifying key phytoconstituents, and lay the groundwork for future research on the biological activities of these calli extracts. Leaf disc and micro shoot tip explants were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BA) and α-naphthaleneacetic acid (NAA) to induce organogenic calli. The resulting calli exhibited distinct biochemical profiles. Moreover, a phytochemical analysis was conducted to compare the metabolite composition of callus 1 (derived from leaf discs) and callus 2 (derived from shoot tips). Callus 1 displayed a higher total phenolic content (30.3558 ± 1.3564 mg (GAE)/g) compared to callus 2 (29.1543 ± 0.9754 mg (GAE)/g). Similarly, callus 1 exhibited a greater total flavonoid content (26.0089 ± 1.8029 mg (RE)/g) than callus 2 (18.4464 ± 1.4797 mg (RE)/g). Liquid chromatography-photodiode array-electrospray ionization-tandem mass spectrometry (LC-PDA-ESI-MS/MS) analysis revealed the presence of 26 and 25 constituents in callus 1 and 2, respectively. Fourteen and thirteen of these identified compounds have been previously reported in other Crocus species, with 22 constituents common to both calli. Twelve constituents were reported here in Crocus for the first time as far as we know.

Indirect shoot regeneration from callus of the shoot tip explants of Gerbera jamesonii Bolus cv. "Festival"

The study was conducted in the Tissue Culture Laboratory, Fadak Agricultural Company, Basrah, Iraq. Mother plants of the “Festival” gerbera cultivar were used for micropropagation by taking the shoot tip explants. The concentration of 2 mgL-1 benzyl adenine (BA) resulted in recording the highest percentage of response to callus induction, reaching 90%, which was significantly superior to the other two treatments, 1 and 1.5mgL-1 BA. The 1 mgL-1 BA treatment was significantly superior in response to indirect shoot generation when compared with the 1.5 and 2 mgL-1 BA treatments. The 1 mgL-1 BA treatment recorded the highest percentage of response to shoot growth induced by callus, which reached 100%. The treatment of callus grown in MS medium supplied with BA concentration of 1 mgL-1 recorded the highest average number of shoots, which reached 10 shoots per 50 mg of callus. The MS medium treatment supplied with a concentration of 0.5 mgL-1 of naphthalene acetic acid (NAA) was significantly superior to the other two treatments of 1 and 1.5 mgL-1 NAA in the percentage of shoot response to root formation. The concentration of 0.5mgL-1 NAA recorded the highest response to root formation, which reached 100%. The treatment of the MS medium supplied with a concentration of 0.5 mgL-1 NAA was significantly superior to the treatments of 1 and 1.5 mgL-1 NAA in the average number of main roots formed on the shoots. This treatment recorded the highest average of main roots, amounting to 3.2 main roots shoot-1. Callus, cultivar, indirect shoot, micropropagation, proliferation, MS salts.

Mass Scale Micropropagation Of Andrographis Paniculata (Burm. F.) Wall Ex Nees

A reliable and efficient protocol was developed for in vitro propagation of Andrographis paniculata (Burm. f.) Wall ex Nees with the use of nodal and shoot tip explants. Nodal segments and shoot apex of in vitro grown seedlings were cultured on agar solidified MS medium supplemented with different concentrations and combinations of plant growth regulators (PGRs). Both the explants underwent direct organogenesis and produced multiple shoot buds. In case of shoot apex, maximum number of multiple shoot buds were formed when cultured on MS medium containing 1.5 mg/l Kn + 1.0 mg/l IAA. But nodal segments produced highest number of shoot buds on MS medium fortified with 1.5 mg/l BAP + 1.0 mg/l IAA. Multiple shoot buds underwent rapid elongation on elongation media and maximum elongation took place on MS medium fortified with 2.0 mg/l Kn + 1.5 mg/l IAA. Elongated shoot buds, on further culture in rooting media, produced strong and stout root systems. Half strength MS medium fortified with 2.0 mg/l IAA was most effective for induction and proliferation of roots. The healthy complete plantlets were successfully acclimatized and established in outside pots through successive phases of acclimatization. The Chittagong Univ. J. B. Sci.,Vol. 7(1 &2):121- 130, 2012.

Harnessing the potential of transverse thin cell layer culture for high-frequency micropropagation of Thai ginseng (Kaempferia parviflora Wall. Ex Baker)

In vitro plant propagation techniques play an important role in modern agriculture and horticulture, offering efficient methods for the accelerated multiplication of high-value plant species. Thin cell layer (TCL) culture technique has emerged as a promising approach for large-scale in vitro plantlets production. This study explores the potential of TCL culture as a cutting-edge strategy to enhance the efficiency of multiple shoots formation in Kaempferia parviflora. An attempt was also made to look into the synergistic effects of cytokinin and auxin [6-benzyladenine (BA) - indole-3-acetic acid (IAA)] during micropropagation. Shoot tip explants (from rhizome sprouts) were cultured onto Murashige and Skoog (MS) medium enriched with BA (2.0–8.0 mg/L) and IAA (0.2–0.8 mg/L) in combination where the highest shoot number (7.3 shoots/explant) and length (5.9 cm) was obtained in 8.0 mg/L BA and 0.8 mg/L IAA. Subsequently, the transverse thin cell layer (tTCL) culture system was successfully utilized whereby the shoot tip explants were individually sliced from tips at the meristem and cultured onto MS medium supplemented with BA (2.0–8.0 mg/L) and IAA (0.2–0.8 mg/L) in combination. The regenerated tTCLs exhibited the highest shoot multiplication (9.3 shoots/tTCL; 28 shoots/shoot tip) and length (5.3 cm) in 4.0 mg/L BA + 0.8 mg/L IAA, thus significantly increased number of plantlets were obtained from tTCL culture (953.0 plantlets) compared to shoot tip culture (527.7 plantlets) after six sub-culture cycles. The tTCL regenerants were successfully acclimatized (with 90% survival) and displayed active growth. Genetic fidelity assessment via inter-simple sequence repeats, conserved DNA-derived polymorphism, directed amplification of minisatellite-region DNA, and start codon targeted polymorphism marker systems suggested that a very low level (5.8%; 148 out of 2572 scorable bands) of polymorphism was detected whereas the ploidy level remained unchanged, confirmed by flow cytometry analysis. The implementation of tTCL culture, coupled with a controlled sub-culture regimen, holds significant potential for the efficient commercial-scale production of K. parviflora.

Propagation of Date Palm (Phoenix dactylifera L.) Cultivar Jawzi Using Shoot Tip

This study reports the successful establishment of a somatic embryogenesis technique for the mass production of a date palm Jawzi cultivar using shoot tip explants. The shoot tip, leaf primordia, and the ground of apical meristems were utilised as explants in the research. The study involved the utilisation of various types of media. It began with initiation media (IM), which had two stages for inducing embryogenesis. This was followed by multiplication media (MM), then elongation shoot media (EM), and finally rooting media (RM) and acclimatization. Embryo induction in the different media types required 50-53 weeks. The first stage, IM2, (3.0 mg.l-l 2iP, 10.0 mg.l-l NAA and 5.0 mg.l-l 2,4-D) for 12 weeks. This was followed by the second stage, IM3, which lasted for 38-41 weeks and included 1.0 mg.l-l BAP, 1.5 mg.l-l 2iP, and 1.0 mg.l-l NAA. These stages enabled us to achieve the optimal value for embryo induction. Afterward the MM3 (0.5. mg.l-l BAP, 0.5 mg.l-l 2iP and 0.5 mg.l-l KIN) showed the highest percentage of total counts of embryo multiplication, while the highest shoot length was attained on EM1 (1.0. mg.l-l BAP, 1.0 mg.l-l KIN and 0.1 mg.l-l IBA). The results also highlighted that RM1 (0.1 mg.l-l BAP, 0.1 mg.l-l KIN,1.0 mg.l-l NAA and 0.5 mg.l-l IBA) showed the highest roots length and roots number, in conclusion, these findings emphasise the importance of media composition in tissue culture protocols. Evaluating the effects of specific media components on different aspects of plant development can optimise tissue culture protocols for plant propagation.

Effect of Nitric Oxide on Browning of Stem Tip Explants of Malus sieversii

Browning is a major problem in the tissue culture of woody plants. Previous studies have shown that nitric oxide (NO) plays a role in regulating plant responses to stress, but its effect on browning in the tissue culture of Malus remains unclear. This study aimed to investigate the impact of exogenous NO donor sodium nitroprusside (SNP) on the browning of Malus sieversii stem tip explants. The results demonstrated that the addition of 50 μM SNP significantly reduced explant browning. Further analysis revealed that exogenous NO decreased the browning index (BD) and levels of malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide anion radical O2−. Additionally, NO treatment increased the activity of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). NO treatment also enhanced the activity of phenylalanine ammonia lyase (PAL), which contributed to the accumulation of flavonoids and inhibited the activity of polyphenol oxidase (PPO) and peroxidase (POD), which are key enzymes involved in the browning process. Furthermore, 50 μM SNP significantly promoted the accumulation of non-enzymatic antioxidants such as proline (Pro), soluble sugar (SS), and soluble protein (SP). Therefore, the results suggest that NO is able to counteract excessive reactive oxygen species (ROS) damage by enhancing both the enzymatic and non-enzymatic antioxidant systems, resulting in a reduction in browning in stem tip explants. Consequently, an improvement in the in vitro propagation efficiency of Malus sieversii shoot tip explants can be achieved.

Using Vitrification–Dehydration Method as a New Protocol for In-vitro Cryopreservation of Date Palm Shoot Tip Cultivars

This study was conducted to develop an in vitro method for the short-term conservation of the planting materials of date palm (Phoenix dactylifera). The conservation of the genetic resources of date palm species and germplasm represented by the cryopreservation of biological material, such as shoot tips of date palm dropped in LN (Liquid Nitrogen) at low temperatures, is a safe method. The main objective of this study was the collection and in vitro germplasm preservation of the date palm cultivars, and this study evaluated the in vitro preservation and genetic stability of date palm shoot tip explants using dimethyl sulfoxide (DMSO) in the medium for long-term storage. Shoot tip explants of about 2-3 cm in length were excised from in vitro cultures and were transferred to preservation media. The results showed that the highest rates of survival (80%) and recovery (75%) were observed with 1.2 M sucrose. To determine the effect of vitrification on freezing tolerances, cultures were exposed to a solution that dissolved the glaze for 60-80 minutes. The maximum survival rate obtained with exposed cultures was 85%. RAPD (randomly amplified polymorphic DNA) was used to explain the differences in the genetic characteristics of cryogenic tissue cultures and non-cryogenic tissues of date palm. Both were similar to the germinated date palm in the open field. Finally, the cryopreserved plants were able to adapt to free-living conditions after acclimatization, All aspects will contribute to the improvement of the currently available techniques for the in vitro germplasm conservation of date palms.

Effect of graphene oxide nanoparticles on in vitro growth of Fragaria x ananassa (Cameron Highlands white Strawberry) and evaluation of genetic stability using DAMD and ISSR markers

Graphene oxide (GO) is a novel nanomaterial with distinct physical properties and significant biological applications. The use of GO in plant tissue culture offers several new properties and potential applications. This research is vital due to the growing need for innovative techniques to promote plant growth, improve plant productivity and mitigate challenges posed by environmental stressors. This study focused on the rare Cameron Highlands white strawberry plants (Fragaria x ananassa) and addressed issues such as callus production during direct shoot induction and hyperhydricity. The research aimed to investigate the effects of GO on the regeneration process and genetic stability of white strawberry plants and to use molecular markers to ensure that plants propagated in vitro are true to type. For this purpose, shoot tip explants were used and different concentrations of GO (0, 2.5, 5.0, 7.5, 10 mg/L) were added to the Murashige and Skoog (MS) medium for six weeks. The results showed that the optimum concentration for promoting the development of white strawberry seedlings was 7.5 mg/L of GO. The study also revealed that the addition of 7.5 mg/L GO in combination with 8 μM TDZ to the MS medium facilitated the induction of multiple shoots. Moreover, the clonal fidelity of the in vitro plants treated with GO showed a genetic similarity of over 97%. These results confirm that lower GO concentrations improve plant development and stability. Consequently, this nanomaterial has a positive effect on the growth of strawberry plants and is therefore well suited for strawberry tissue culture.

Micro propagation and evaluation of growth parameters of Bacopa monnieri (L)

Micro propagation and evaluation of growth parameters of Bacopa monnieri (L)

Cryopreservation of Lilium Candidum Germplasm: Analysis of Pre- and Post-freeze Treatments

BACKGROUND: Lilium candidum L. is a perennial ornamental plant that has various medicinal properties and is used in the cosmetic industry. The species is facing threats from urbanization and climate change and requires urgent protection. The most secure and efficient technology for the longterm storage of plant genetic resources is cryopreservation, which involves preserving genetic material at extremely low temperatures. OBJECTIVE: Today, plant biodiversity is endangered because of the narrowing of its natural distribution areas and/or destruction for different purposes. This study concentrated on creating a cryopreservation process using shoot tips and calluses as explant sources for the long-term conservation of L. candidum species. MATERIALS AND METHODS: Populations of L. candidum naturally distributed from three different regions of Turkey (Kepsut, Balikesir; the area surrounding Bafa Lake, Aydın ; and Fethiye- Mu la) were grown in vitro to supply shoot tip and callus explants. Prior to freezing by droplet-vitrification and vitrification techniques, shoot tips and calluses were treated with MS nutritional medium supplemented with 0.4 M sucrose 7 g/L agar and plant vitrification solution 2 (PVS2). RESULTS: Cryopreserved shoot tips showed the highest levels of regeneration (71.8%) after a PVS2 treatment of 90 min, while calluses showed the highest levels of regrowth (63.9%) after a PVS2 exposure of 60 min. CONCLUSION: High levels of regrowth are produced when the various cryopreservation procedures described here are used to preserve both shoot tip and callus explants. This potentially makes the method promising for the long-term preservation of endangered L. candidum varieties.

Plant Growth Regulator- and Elicitor-Mediated Enhancement of Biomass and Andrographolide Production of Shoot Tip-Culture-Derived Plantlets of Andrographis paniculata (Burm.f.) Wall. (Hempedu Bumi).

Andrographis paniculata (Burm.f.) Wall. (Acanthaceae) is revered for its medicinal properties. In vitro culture of medicinal plants has assisted in improving both the quantity and quality of their yield. The current study investigated the effects of different surface sterilization treatments, plant growth regulators (PGRs), and elicitors on culture establishment and axillary shoot multiplication of A. paniculata. Subsequently, the production of andrographolide in the in vitro plantlets was evaluated using high-performance liquid chromatography (HPLC) analysis. The shoot-tip explant was successfully sterilized using 60% commercial bleach for 5 min of immersion with a 90% survival rate and 96.67% aseptic culture. The optimal PGR for shoot growth was 6-benzylaminopurine (BAP) at 17.76 µM, supplemented into Murashige and Skoog (MS) media, producing 23.57 ± 0.48 leaves, 7.33 ± 0.10 shoots, and a 3.06 ± 0.02 cm length of shoots. Subsequently, MS medium supplemented with 5 mg/L chitosan produced 26.07 ± 0.14 leaves, 8.33 ± 0.07 shoots, and a 3.63 ± 0.02 cm length of shoots. The highest andrographolide content was obtained using the plantlets harvested from 5 mg/L chitosan with 2463.03 ± 0.398 µg/mL compared to the control (without elicitation) with 256.73 ± 0.341 µg/mL (859.39% increase). The results imply that the protocol for the shoot-tip culture of A. paniculata was developed, and that elicitation enhanced the herbage yield and the production of andrographolide.

Establishment of In Vitro Cultures and Investigation of Micropropagation Possibilities in Shoot Tip Explants of Different Mastic Tree (Pistacia lentiscus L.) Genotypes

Establishment of In Vitro Cultures and Investigation of Micropropagation Possibilities in Shoot Tip Explants of Different Mastic Tree (Pistacia lentiscus L.) Genotypes

Employment of In Vitro Technique to Propagate Stevia (Stevia rebaudiana) Plant

The current study was conducted with aim to find an efficient simple protocol for propagation of stevia plant ( Stevia rebaudiana ) In Vitro. Shoot tips, leaves and stem nodes explants were sterilized with different concentrations of NaOCl (0.0, 2.0, 4.0 and 6% v/v) then cultured on MS medium supplemented with 30 g.l-1 sucrose. Results revealed that, no contamination was recorded when using 4.0 % and 6.0 % of NaOCl with (100% and 8.9%) survival at 4.0% and 6.0 % respectively. Moreover, combination of plant growth regulators which were used in this study, have been successfully optimized. However, best combination for number of shoots. explant-1 was found to be (0.4 BAP + 0.2 Kin mg.l-1) for shoot tips explants with an average of 16.44 while (2.0 BAP + 0.25 GA3 mg. l-1) produced maximum average of plant height from shoot tips reached 4.85 cm. For in vitro roots induction, maximum root induction, number and length of roots. Shootlets reached (93.3 %, 9.77 and 9.0 cm) respectively when cultured shootlets on MS medium supplemented with 2.0 mg.l-1 of IBA.

Plant fruit extracts enhance the in vitro propagation of cowpea (Vigna unguiculata) on Murashige and Skoog media

Cowpea (Vigna unguiculata) is a versatile legume with diverse nutritional and nutraceutical properties that serve as a food security and medicinal crop for millions of households across Africa. An efficient protocol was developed to propagate shoot tip and cotyledonary node explants from six cowpea breeding accessions in vitro on Murashige and Skoog (MS) basal media supplemented with either banana extract, coconut water, orange or tomato juice. Micropropagation performance was compared to MS medium supplemented with B5 vitamins. A total of 500 plantlets were obtained in vitro across treatments and MS basal media supplemented with tomato juice had the highest micropropagation performance (154 plantlets), followed by banana extract (112 plantlets), orange juice (107 plantlets), and coconut water (82 plantlets). Three accessions (AGRAC 216, TA, and Asontem) were found to be the most amenable to in vitro propagation using plant-derived extracts. Overall, this study successfully established that plant-derived extracts can support in vitro cowpea propagation in the absence of synthetic plant growth regulators.

Effect of BAP (6-Benzylaminopurine) and NAA (α-Napthalene Acetic Acid) treatment on Micropropagation of Adansonia digitata L.

The African Baobab, also known as Kalpvriksha (Adansonia digitata L.), is a significant multi-purpose tree that has gained popularity among people, primarily in African countries, for its use as food, fodder, clothing, and ropes. There is one of the obstacle arises in growing this plant by farmers in India is the lack of planting materials. The young plants of A. digitata L. have to be imported form selected nurseries. Therefore, micropropagation can be used as a substitute method to create offspring that are genetically similar to the parent plant. In order to micropropagate Kalpvriksh (A. digitata L.), this experiment is being conducted utilizing shoot tip explants. For shoot proliferation, these explants are grown in MS medium containing various concentrations of BAP (6- Benzylamino purine): 0.5, 1.0, 1.5, 2.5, 5.0 mg/L. Shoots are rooted in a medium containing at the same concentration of NAA (α-Napthalene Acetic Acid) 0.5 mg/L alone or with combination. Complete Randomized Design (CRD) is used to conduct each experiment. ANOVA and the Duncan New Multiple Range test are used to examine the data. According to the technique for micropropagation of A. digitata L. The concentration of BAP and NAA was found to be 1.5 mg/L and 0.5 mg/L respectively which showed better enhancement for shoot regeneration and rooting for micropropagation of Kalpvriksha (Adansonia digitata L.) tree.

In vitro direct organogenesis of the medicinal single-mountain local prioritized vulnerable Greek endemic Achillea occulta under different medium variants

Achillea species are of high medicinal value based on their use in traditional medicine and their content in secondary metabolites with potential application in dermatology and cosmetic industry. Achillea occulta is a single-mountain local prioritized vulnerable Greek endemic encountering propagation problems through seeds and root division. In vitro propagation can be used for the rapid and mass production of clonal plants complying with phytochemical and sanitary quality criteria. In this study, several culture medium variants were tested including the effect of different indole-3-butyric acid (IBA) concentrations with 6-benzyladenine (BA), different cytokinin types, agar concentrations, and zeatin (ZT) sterilization methods on in vitro shoot proliferation, as well as the effect of different auxin types and concentrations on in vitro rooting and ex vitro survival rate of shoot-tip explants. The results showed BA was the most preferable cytokinin type as evidenced by all proliferation attributes and autoclaved ZT provided longer shoots. Therefore, the two more promising and cost-effective treatments for proliferation were 2.2 μM BA + 0.25 μM IBA or 4.4 μM BA + 0.5 μM IBA after 21 days of culture on modified Murashige-Skoog (MS) medium (x2 Fe) enriched with 20 g L-1 sucrose and 6-7 g L-1 Agar (90-93.8% shoot formation, 2.7-2.9 shoots 0.4-0.6 cm long, 2.5-2.6 proliferation rates). Explants rooted better after 21 days of culture on plain MS medium with 2 μM IBA or 1 μM α-naphthalene acetic acid (NAA) (13.3-20% rooting, 1-1.5 roots 1.8-2 cm long). NAA gave more roots, IBA longer roots, while both auxins exhibited highest rooting (13.33-20%) and ex vitro survival rates (50-66.7%).

Proline and Silver Nitrate Promotes Multiple Shoot Induction from Mature Embryo and Shoot Tip Explants of Sorghum

Proline and Silver Nitrate Promotes Multiple Shoot Induction from Mature Embryo and Shoot Tip Explants of Sorghum

Effect of Medium Components on in vitro Shoot Formation and Rooting of Papaya (Carica papaya L.)

The effect of modification of some selected MS nutrientmedium on in vitro growth and development of papaya (Carica papayaL.) shoot tips was examined with the objective of optimizing shootregeneration and rooting efficiencies. Modifications were made onselected components of MS medium. Shoot tip explants were obtainedfrom in vitro-grown plantlets. The results showed that MS salts at thetwice full strength was optimal for all parameters measured comparedwith other strengths tested. Sucrose concentrations of 0.75% and 1.5%were better than the other concentrations tested for growth anddevelopment of cultured shoot tips, and there was no significantdifference between them. The results revealed the importance of inclusionof BA and NAA in the nutrient medium at relatively low concentrations,the best results for shoot and leaf formation was recorded with thecombination of BA at 1.0 mg /litre and NAA at 0.01mg /litre, where thegreatest number of shoots and leaves was obtained. The combinationtreatment of 0.3 mg/litre BA and 0.00 mg/litre NAA recorded the bestresults for mean shoot elongation, compared with the other combinationtreatments tested. However, the produced roots were morphologicallyabnormal. The study attested to the essentiality of IBA for rooting of invitro produced papaya shoots, and the data suggested the conduction ofadditional studies for determination of optimal concentration for rooting

Protocol for in vitro regeneration and rapid mass multiplication of apetalous male sterile lines of marigold

The apetalous form of male sterility, controlled by a single recessive gene (msms), is widely used for F1 hybridseed production in marigold (Tagetes spp.). Maintenance of this form of male sterility through conventionalmethods is a highly labour-intensive and costly task. Hence, we have developed a viable in vitro regenerationprotocol for rapid multiplication in three apetalous male sterile lines of marigold, MS-5, MS-7, and MS-8. Theeffect of Thidiazuron (TDZ) was assessed on in vitro establishment of apetalous male sterile lines using shoottipexplants. The modified MSm medium supplemented with 1.5 mg/l TDZ and 0.2 mg/l NAA was found suitablefor culture establishment from shoot tip explants. We have also assessed the efficiency of two cytokinins,TDZ and Kinetin (KIN), for rapid multiplication of male sterile lines of marigold. The culture media, genotypesand their interactions significantly influenced the shoot proliferation of marigold. Our findings have revealedthe superiority of kinetin over TDZ in inducing quality shoots during in vitro proliferation phase. The highestproliferation of quality shoots was obtained on modified MSm medium enriched with 0.5 mg/l kinetin and 0.1mg/l NAA. Half-strength MS medium supplemented with 0.5 mg/l of IBA was found effective for the inductionof quality roots in all the tested genotypes of marigold. Among the three male sterile lines of marigold, MS-5exhibited better in vitro establishment, proliferation and rooting abilities over the MS-7 and MS-8 lines. Thedeveloped protocol can be efficiently utilized for rapid in vitro mass multiplication of apetalous male sterilelines of marigold.