Number Of Multiple Shoots Research Articles

In vitro plantlet regeneration from cotyledon explants of Capsicum annuum L

An efficient In vitro plant regeneration was established in Capsicum annuum L. from cotyledon explants. The best performance i.e., 80% of the explants responded for multiple shoot bud proliferation on MS medium containing 10 mg/l Zeatin. The maximum number of buds per explants was 50.28 ± 2.69 and the bud length of 0.5 ± 0.22 cm was obtained in this medium after 30 days. The average number of 80.32 ± 0.16 multiple shoot bud and the average shoot bud length of 1.0 ± 0.19 cm was attained on the same medium after 90 days. These shoot buds on transfer to MS + 10 mg/l Zeatin + 2.0 mg/l GA3 yielded 6.3 ± 0.35 number of multiple shoots with an average shoot length of 4.6 ± 0.62 cm after 30 days. Shoots rooted well (80% of induced shoots) on half strength MS medium supplemented with 0.5 mg/l IBA. The average number of roots per shoot was 8.2 ± 0.79 and the average root length of 4.5 ± 0.58 cm was recorded. About 80% of In vitro derived plantlets were survived under open field conditions.

IN-VITRO REGENERATION OF MULTIPLE SHOOTS FROM APICAL MERISTEMS OF COWPEA (Vigna unguiculata L. (WALP) USING DIFFERENT GENOTYPES (LOCAL AND IMPROVED) AND MEDIA COMPOSITIONS

Organogenesis in-vitro was evaluated in explants derived from shoot apices of four cowpea varieties cultured on Murashige and Skoog (MS) basal medium. To evaluate the response of the cowpea genotypes to in vitro regeneration, seedlings were drived from zygotic embryos excised from surface sterilized seeds after two weeks of culture on MS basal medium + vitamins supplement with 30% sucrose. To regenerate multiple shoots, apices of the in vitro seedlings with average length of 1cm were excised and cultured on MS supplemented with 0.5, 1.0 or 1.5mg/L 6-Benzylamino purine(BAP) with or without 0.1mg/L α-naphthaleneacetic acid (NAA) for six weeks. The highest shoot number was recorded with IT99k-573-2-1 and Danilla and shoot length was highest in Kanannado, IT04k-33-2-1and Danilla indicating that Danilla responded well to in vitro regeneration. To optimise the production of multiple shoots the effects of different concentratios (0.5, 1.0 and 1.5mg/L) of 6-benzyaminopurine (BAP) with or without 0.1mg/L α-naphthaleneacetic acid on the number of responding explant, number of multiple shoot produced and length of the shoots were tested. after six weeks of culture, the percentage of responding shoot apices was highest on media supplemented with 1.5mg/L BAP with and without 0.1mg/L NAA.The number of responding apices decreasing with decrease in the concentration of BAP. Maximum number of multiple shoots and the highest mean shoot length were also obtained when MS was supplemented with 1.5mg/L BAP with and without 0.1mg/L NAA.

Micropropagation of diploid and tetraploid Plectranthus amboinicus (Lour.) Spreng

Plectranthus amboinicus (Lour.) Spreng is commonly consumed by people in North Sumatra, Indonesia, as a vegetable, which is commonly called Torbangun. Plant tissue culture is an alternative method for the mass-production of transplants. Genetic improvement by somatic cell manipulation such as induction of polyploid plants is needed to improve biomass production as well as their secondary metabolite products. This research aimed to conduct in vitro micropropagation of diploid and tetraploid P. amboinicus on Murashige and Skoog (MS) medium containing BAP or Kinetin. Shoot tips were used as explants and cultured on MS medium containing BAP or Kinetin at 0, 0.5, 1 and 2 mg/l given as single concentration. The experiment used a completely randomized design with four replications. Each replication consisted of three explants. The results showed that height of shoots was affected by type of cytokinins. Kinetin at 0.5 mg/l gave the highest shoots of tetraploid plants significantly different with addition of BAP both to diploid and tetraploid plants. The number of multiple shoots and leaves varied in both diploid and tetraploid plants. Root formation was best on the medium without addition of cytokinins. Both diploid and tetraploid plantlets had a high survival rate after acclimatization in the greenhouse.

Salinity Effects on Gene Expression, Morphological, and Physio-Biochemical Responses of Stevia rebaudiana Bertoni In Vitro.

Stevia rebaudiana Bertoni is a little bush, which is cultivated on a large scale in many countries for medicinal purposes and used as a natural sweetener in food products. The present work aims to conduct a protocol for stevia propagation in vitro to produce and introduce Stevia rebaudiana plants as a new sweetener crop to Egyptian agriculture. To efficiently maximize its propagation, it is important to study the influence of stress factors on the growth and development of Stevia rebaudiana grown in vitro. Two stevia varieties were investigated (Sugar High A3 and Spanti) against salt stress. Leaves were used as the source of explants for callus initiation, regeneration, multiplication and rooting. Some stress-related traits, i.e., photosynthetic pigments, proline contents, and enzyme activity for peroxidase (POD), polyphenol oxidase (PPO), and malate dehydrogenase (MDH) were studied. Murashig and Skoog (MS) medium was supplemented with four NaCl concentrations: 500, 1000, 2000, and 3000 mgL−1, while a salt-free medium was used as the control. The data revealed that salinity negatively affected all studied characters: the number of surviving calli, regeneration%, shoot length, the number of multiple shoots, number of leaf plantlets−1, number of root plantlets−1, and root length. The data also revealed that Sugar High A3 is more tolerant than Spanti. The total chlorophyll content decreased gradually with increasing NaCl concentration. However, the opposite was true for proline content. Isozyme’s fractionation exhibited high levels of variability among the two varieties. Various biochemical parameters associated with salt tolerance were detected in POD. Namely, POD4, POD6, POD 9 at an Rf of 0.34, 0.57, and 0.91 in the Sugar High A3 variety under high salt concentration conditions, as well as POD 10 at an Rf of 0.98 in both varieties under high salt concentrations. In addition, the overexpression of POD 5 and POD 10 at Rf 0.52 and 0.83 was found in both varieties at high NaCl concentrations. Biochemical parameters associated with salt tolerance were detected in PPO (PPO1, PPO2 and PPO4 at an Rf of 0.38, 0.42 and 0.62 in the Sugar High A3 variety under high salt concentrations) and MDH (MDH 3 at an Rf of 0.40 in both varieties at high salt concentrations). Therefore, these could be considered as important biochemical markers associated with salt tolerance and could be applied in stevia breeding programs (marker-assisted selection). This investigation recommends stevia variety Sugar High A3 to be cultivated under salt conditions.

Invitro germination of Bael (Aegle marmelos (L.) Corr.) seeds for clonal propagation

Aegle marmelos, commonly known as Bael, is one of the most important medicinal plants of Indian tradition. All parts of the tree viz. leaf, fruit, bark and root have medicinal properties and have been used in many traditional medicine systems. The present study trial has been taken up at Horticultural College & Research Institute, Coimbatore to establish a protocol for in vitro germination of seeds to use it further for clonal propagation. The investigation revealed that the seeds without seed coat gave more number of multiple shoots (5.57) where as the seeds with seed coat gave single seedling. The days taken for germination was lesser in seeds without seed coat (14.76 days) than the the seeds with seed coat (20.59 days). The longest seedling was observed in the seeds with seed coat (2.09 cm) compared to that of seeds without seed coat (1.22 cm). the days taken for budbreak, number of shoots were higher in the MS medium supplemented with BAP 1.5 mgl-1. Comparison between the explants from in vitro and field grown seedlings showed that the time taken for culture response was much earlier in the in vitro derived seedlings. All the explants showed better response in the basal MS medium supplemented with BAP 1.5 mgl-1

In vitro Regeneration and Proliferation of Maize (Zea mays L.) Genotypes through Direct Organogenesis

The variability of in vitro regeneration and proliferation of maize genotypes POOLISSRQPMX, DTSR-WCO, TZLCOMP4C3, TZE COMP 3C2, EV99QPM, POP66 SR/ACR94-YQPM, DTSR-WC, SAMMAZ 19S-14DT and TZEE-YPOPSTRC4 was investigated. Explants were regenerated through direct organogenesis using 0.1 mg/l NAA + 2.0 mg/l BAP, 0.1 mg/l NAA + 2.0 mg/l KIN, 0.3 mg/l NAA + 3.0 mg/l BAP, 0.3 mg/l NAA + 3.0 mg/l KIN, 0.5 mg/l NAA + 4.0 mg/l BAP, 0.5 mg/l NAA + 4.0 mg/l KIN and MS medium only. The regenerated maize yielded the highest number of multiple shoots (1.70 cm) and shoot length (3.35 cm) on MS medium supplemented with 0.3 mg/l NAA + 3.0 mg/l BAP within 31 days. Genotype EV99QPM and DTSR-WCO had the highest number of shoots, rooting traits respectively. Keywords: Maize, regeneration, hormones, variability, survival, character DOI : 10.7176/JNSR/9-6-09 Publication date :March 31 st 2019

UV-C irradiation induced alterations in shoot proliferation and in vitro flowering in plantlets developed from encapsulated and non-encapsulated microshoots of Persian violet

Abstract Ultraviolet-C (UV-C) radiation is well-known for its germicidal effect but its mutagenic potential in aiding new plant variety development has not been explored extensively. Persian violet (Exacum affine Balf.f. ex Regel) is a well-known decorative plant in Thailand. In this research, the effects of 0–4 h of UV-C irradiation on shoot proliferation and in vitro flowering of Persian violet plantlets derived from non-encapsulated and encapsulated microshoots were investigated. The number of multiple shoots was found to decrease as UV-C exposure time increased. Shoot-tip decapitation increased floral number in plantlets derived from both non-capsulated and encapsulated microshoots. The plantlets developed from non-encapsulated microshoots exposed to 4 h of UV-C irradiation exhibited tiny flower buds within 3 days from shoot-tip decapitation of the plantlets. However, the time for flower initiation was 35 days in control plantlets developed from non-encapsulated microshoots without irradiation. Another interesting novel finding is that 4 h of UV-C irradiation of the non-encapsulated microshoots resulted in the highest number of flowers (6.80 flowers) in the plantlets after 10 weeks compared to all the other treatments including the control that had only 3.20 flowers. This finding of Persian violet flowers with 7 petals has never been reported before. However, this UV-C treatment of the non-encapsulated microshoots diminished viability and in vitro germinability of pollen more than those in the treatment of encapsulated microshoots. Thus, UV-C irradiation of Persian violet microshoots affected multiple shoot proliferation and in vitro flowering. Encapsulation of the microshoots could counteract many of these effects of UV-C irradiation.

Role of Phytohormones and PGR's in Micropropagation of Anethum graveolens L. through Axillary Shoots and Evaluation of NaCl effect on Growth Parameters

An In vitro protocol is standardized for rapid in vitro propagation of Anethum graveolens through enhanced axillary shoot induction and optimization of role of BAP on shoot proliferation. MS basal medium augmented with various hormones (BA, NAA, KIN, and IBA) was used for multiple shoot development and rhizogenic response. The influence of various concentrations of NaCl was also observed on plant growth and morphological parameters. MS media fortified with BA (0.5–1.5) singly was induce axillary bud proliferation and in combination with NAA (0.1–0.25 mg L−1) and KIN (0.25–0.50 mg L−1), it was able to induce multiple shoots. BA at 1.0 mg L−1, KIN 0.5 mg L−1 and NAA 0.25 mg L−1 induced optimum number of multiple shoots (11.00 ± 0.379). The maximum frequency of rooting 83% with 3.67 ± 0.66 roots per explants was recorded on half strength MS medium supplemented with 1/2 MS + IBA 1.0 mg L−1 within 4 weeks of transfer of the shoots to the rooting medium. Hardening was successfully attained under partially controlled conditions inside the hardening room. The method could effectively used for conservation and clonal propagation to meet the pharmaceutical demands of this medicinally important plant species.

Role of silver nitrate in plant regeneration from cotyledonary nodal segment explants of Prosopis cineraria (L.) Druce.: A recalcitrant medicinal leguminous tree

Abstract An efficient protocol for in vitro plant regeneration from cotyledonary nodal segment explants of Prosopis cineraria (L.) Druce., a multipurpose leguminous tree, was established. Nodal explants were excised from 5-day-old seedlings and cultured on Murashige and Skoog (MS) medium fortified with different concentrations of BAP (0.44–2.22 µmol/l) alone for shoot bud regeneration. Highest percent of shoot bud regeneration (87.66%) was noticed on MS medium supplemented with 2.22 µmol/l BAP. The nodal segments derived from seedlings were placed on MS medium with different concentrations of BAP (0.44–2.22 µmol/l) in combination with 0.46 µmol/l for shoot bud multiplication. The highest percent of shoot bud multiplication (93.50%) with 3.5 shoots/explant was recorded on MS medium containing 2.22 µmol/l BAP and 0.46 µmol/l KIN combination. The number of multiple shoots was further increased to 12.0 shoots/culture when the combination of 2.22 µmol/l BAP, 0.46 µmol/l KIN and 0.59 µmol/l AgNO3 was used. For rooting, the elongated shoots (>2 cm) were cultured on MS medium augmented with various concentrations of NAA (0.53–2.68 µmol/l) along with 0.46 µmol/l KIN and 0.59 µmol/l AgNO3. Among the combinations tested, the highest percent of root formation (87.66%) with 4 roots/shoot was observed in the medium containing 0.53 µmol/l NAA, 0.46 µmol/l KIN and 0.59 µmol/l AgNO3 combination. Rooted plantlets were successfully transferred into plastic cups and acclimatized under greenhouse conditions. Subsequently, they were established in the field and grew normally.

In vitro propagation of Rhododendron macabeanum Watt ex Balf.f., an endangered and endemic Rhododendron species from Manipur and Nagaland, India

An efficient and reproducible protocol for in vitro propagation of R. macabeanum, an endemic and endangered species from North East India was carried out successfully. Both ex vivo and in vitro seed germination was studied. Effect of activated charcoal (0.2%) in the Anderson medium (AM) was studied for seed germination but higher germination was obtained on basal medium alone. Different explants viz., apical shoots, nodal and root segments from in vitro raised seedlings were used for multiple shoot induction. Out of the three cytokinins studied in AM for multiple shoot induction, 2-isopentenyladenine (2iP) was the most responsive with optimum concentration at 19.68 µM following which experiments were also carried out with 2iP in different basal media viz., AM, modified Anderson medium and woody plant medium (WPM). The number of multiple shoots produced was higher and healthier in WPM with 8.15 ± 0.20a shoots per explant and an average shoot length of 2.13 ± 0.02a cm. 100% in vitro rooting was attained in WPM with 0.2% activated charcoal and successfully transferred from lab to land with about 90% survival rate.

Establishment of an efficient in vitro regeneration and Agrobacterium rhizogenes-mediated genetic transformation protocol for safed musli (Chlorophytum borivilianum Santapau & R.R.Fern.)

Safed musli (Chlorophytum borivilianum Santapau & R.R.Fern.) is a medicinal herb valued for its tuberous roots that contain several bioactive compounds. Conventionally, safed musli tubers are harvested for secondary metabolites 9 mo after planting. Alternatively, in vitro culture can be used for mass production and to shorten the time required to produce secondary metabolites. This study was designed to develop an efficient hairy root induction protocol for safed musli. Shoot bud explants of various lengths were tested on Murashige and Skoog medium with 3 mg L−1 6-benzylaminopurine. Shoot buds >4 cm resulted in 100% multiplication, but 2- to 4-cm shoot buds produced a high multiplication rate (92%), the highest number of multiple shoots (15.55 ± 0.37), and the longest shoots (6.83 ± 0.08 cm) after 30 d of culture. In vitro shoots were infected with one of five strains of Agrobacterium rhizogenes. Only strains MAFF106590, MAFF106591, and MAFF720002 induced the hairy root phenotype indicative of genetic transformation. MAFF720002 strain had a positive transformation response in all in vitro raised safed musli stem segments of different lengths. Stems 2.5 cm in length had the highest transformation percentage (76 to 81%), greatest number of hairy roots per explant (5.02 ± 0.06), and the longest hairy roots (23 ± 0.49 cm). PCR analysis detected the rolB gene in the transformed samples. The A. rhizogenes strain played a significant role in producing and promoting the growth of hairy roots in safed musli. This approach can potentially be utilized for the replacement of conventional field methods for secondary metabolite production from safed musli.

In Vitro Direct Regeneration through Cotyledon Culture in Pigeon Pea [Cajanus Cajan (L.) Millsp.] and Evaluation of Genetic Fidelity using RAPD Markers

In Vitro Direct Regeneration through Cotyledon Culture in Pigeon Pea [Cajanus Cajan (L.) Millsp.] and Evaluation of Genetic Fidelity using RAPD Markers An efficient and direct in vitro regeneration of Cajanus cajan (red gram) cv ICP 26 from cotyledon explants was established. Cotyledons of 7-8 day old in vitro grown seedlings were cultured on MS/B5 medium augmented with different concentrations (0.5-5.0 mg/L) of 6-benzylaminopurine (BAP) / Kinetin (KIN). Among these concentrations, 2.0 mg/L BAP was found to be efficient in inducing maximum number of multiple shoots with 80% of response. 80% of the elongated shoots were successfully rooted on MS medium fortified with 1.0 mg/L indole-3-butyric acid (IBA). These in vitro rooted plantlets were successfully established in soil where 80- 90% of them developed into normal and fertile plants. 20 RAPD primers were used to assess the genetic fidelity of randomly selected regenerated plantlets along with the donar plant. Out of 20 RAPD primers screened only 7 primers produced clear, distinct and scorable bands with an average of 2.5 bands per primer. All banding profiles from regenerated plantlets were monomorphic and similar to those of the mother plant. The amplification pattern confirmed the genetic uniformity of the regenerated plantlets. Thus, this reproducible regeneration protocol can be used for Agrobacterium tumefaciens mediated genetic transformation in pigeon pea....

In Vitro Regeneration of Pigeonpea [Cajanus cajan (L.) Millsp.] Genotype GT 101 using Cotyledonary Node

Objectives: A protocol was developed for plant regeneration using cotyledonary node of high-yielding indigenous pigeonpea [Cajanus cajan (L.) Millsp.] genotype GT 101. Methods: Induction of multiple shoots directly was completed from cotyledonary node as an explants on Murashige and Skoog’s (MS) medium supplemented with various concentrations and combination of 6-benzylaminopurine (BAP), kinetin and α-naphthalene acetic acid (NAA). Elongation of multiplied shoots was performed on MS medium supplemented with different combination of BAP and GA3. These well elongated plantlets were further transferred on MS medium supplemented with various concentrations of indole-3-butyric acid (IBA) for root induction. Regenerated plants were transferred to cocopeat:soil:vermiculite (2:1:2) for acclimation. Findings: The frequency of multiplication and number of multiple shoots from cotyledonary node explant were influenced on various types and concentrations of cytokinin. For multiple shoots induction, 3.0 mg/L BAP with 0.5 mg/L NAA was superior as compared to other combinations. The elongation of multiplied shoots was carried out on MS medium supplemented with 0.5 mg/L BAP and 0.5 mg/L GA3. The developed shoots were advanced to rooting on the medium supplemented with 0.5 mg/L IBA. They were subsequently grown in pots with 80% survival rate and these plants produced viable seeds. Improvement: The protocol for the production of in vitro multiple shoots with high frequency and their successive conversion to whole plants agreements potential for use in the improvement of protocol for development of transgenic in pigeonpea.

English

Melia dubia Cav. (Meliaceae) is a multipurpose tree of tropical and subtropical regions mainly cultivated for its medicinal and industrial importance. Due to its versatile properties, it has been depleted in its natural environment. Moreover due to sluggish and poor seed germination, there is a threat of its gene pool exclusion from the natural habitat. The alternative method for conservation and efficient mass propagation is thus need of the hour. As per the extensive literature survey there is no report on efficient protocol for mass propagation of M. dubia through callus organogenesis. Therefore, the present work was aimed to develop in vitro organogenesis protocol for rapid and large scale production of planting material. From our results, maximum callus percentage, callus weight and fragile callus was observed on 1.0 mg/l benzylaminopurine (BAP) in combination with 0.5 mg/l naphthalene acetic acid (NAA). The callus differentiation was achieved at different concentrations of BAP and indole acetic acid (IAA). Multiple Shoot number per callus propagule 5.30 was observed on 0.5 mg/l BAP and 1 mg/l IAA concentration. The maximum rooting percentage (78.5%), root number per explant (4.33) and root length per explant (4.41 cm) was observed at 0.5 mg/l indol butyric acid (IBA) after 30 days of inoculation. Further the total flavonoid content, phenolic content and antioxidant properties of leaves of in-vitro regenerated plants where studied. Total flavonoids and phenolic content in leaves of in vitro Melia dubia was 0.56 ± 0.8 mg quercitin equivalent (QE) and 2.97 ± 0.17 mg gallic acid equivalent (GAE) respectively. The antioxidant property was further assed through measurement of DPPH radical scavenging activity. The in-vitro regeneration protocol can be exploited for commercial cultivation and fulfilling the growing demand for fresh explant material through mass propagation of M. dubia an economically important plant species. Key words:  Melia dubia, antioxidant, indole-3-butyric acid, flavonoids and phenolics.

Influence of Ethylene Inhibitor Silver Nitrate on Direct Shoot Regeneration from <i>in Vitro</i> Raised Shoot Tip Explants of <i>Sphaeranthus indicus</i> Linn.—An Important Antijaundice Medicinal Plant

In the present investigation, an attempt has been made to study the influence of ethylene inhibitor silver nitrate on direct shoot regeneration in Sphaeranthus indicus, an important antijaundice medicinal plant, by using in vitro raised shoot tip explants. The effect of various concentrations of kinetin, BAP (0.5 - 3.0 mg/l), and NAA (0.1 - 0.5 mg/l) along with AgNO3 (0.1 - 1.0 mg/l) was studied. Among the combinations tested MS medium augmented with kinetin (1.0 mg/l), NAA (0.1 mg/l) and AgNO3 (0.4 mg/l) was found to be optimum for production of multiple shoots (34.3 ± 0.36). Addition of AgNO3 to the media not only increases shoot number in all the concentrations tested but also shoot length. AgNO3 at the concentration of 0.4 mg/l produced 35% more number of multiple shoots when compared to multiple shoots (10.8 ± 0.12) produced in control. In the present study by the addition of ethylene inhibitor silver nitrate and growth regulators, more number of multiple shoots (three folds) and shoot length was observed compared to control. These in vitro raised shoots were transferred to the rooting medium containing different concentrations of auxins such as NAA and IAA along with AgNO3 (0.1 - 0.6 mg/l). Better rooting response (21.6) was observed on NAA (2.0 mg/l) and AgNO3 (0.4 mg/l) containing media. The healthy rooted plantlets were transferred to polybags containing soil and vermiculate in 1:1 ratio for hardening. Finally the hardened plants were transferred to field environment for utmost survivability.

Micropropagation and Attempts to Develop RAPD Molecular Markers for Salt Tolerant Cultivars of Pearl Millet

Differences were observed among four salt tolerant cultivars of pearl millet to in vitro culture media. Among the four cultivars, Cv IP 6098 is the most salt sensitive had maximum number of multiple shoots (15)/ seed, and the more salt tolerant cvs IP 6106 and IP 22269 developed 3-6 shoots/seed while cv IP 19612 the seeds remained single shooted in MS+3.0mg/L media up to 7 months. The multiple shoots 20% of cv IP 6098, flowered in 5 months with the reduction in hormone concentration (MS+1.0 mg/L BAP) whereas, the other three cultivars did not flower under the tested conditions.
 The present study was carried to detect genetic differences among the four cultivars of pearl millet at molecular level through RAPD PCR markers. RAPD analysis was carried with 12 primers, each 10-mer, only 7 primers were amplified showed polymorphism for the four cultivars. Primer OPM 16 revealed a single unique band of approx. 485bp in the genotype IP 22269 and three polymorphic bands in the genotype IP 6106. The results were repeatable, and further studies in this direction may enable to link the single band in the genotype IP 22269 to a morphological trait or physiological condition like salt tolerance.

English

The present attempt aimed to optimize micropropagation protocols supplemented with different concentrations and combinations of benzylaminopurine (BAP) (0, 2, 4, 6 mg L-1) and indole acetic acid (IAA) (0.5 and 1.0 mg L-1). Exotic banana (Musa spp) genotypes GCTCV-215 (AAA), ‘Yangambi’ Yangambi Km-5 (AAA) and FHIA-23 (AAAA) were used in research work. Experiments were conducted at Plant Tissue Culture Laboratory of Nuclear Institute of Agriculture (NIA), Tando Jam. Data collected for in vitro shoot consists of the following parameters: days for bud initiation, rate of shoot proliferation (%), number of multiple shoots, shoot length (cm) and fresh mass of shoot (g). Significant (p≤ 0.05) variations were observed for varieties, treatments and varieties x treatment for all the parameters. Synergistic effects of BAP and IAA were observed in GCTCV-215 and Yangambi Km-5. Out of various treatments, best concentration for multiple shoot in short period of time for GCTCV-215 and Yangambi Km-5 was found in 4.0 mg /l BAP + 0.5 mg L-1 IAA. Maximum fresh mass of shoot observed at same concentration and combination of BAP and IAA and for shoot length combination of 4.0 mg L-1 BAP with 1.0 mg L-1 IAA was found to be most suitable for GCTCV-215 and Yangambi Km-5. FHIA-23, show better performance in MS medium supplemented with only BAP at concentration 4.0 mg/L-1. After development of root, in vitro plantlets were shifted from growth room to green house in polythene bags containing garden soil and humus mixture in ratio (1:1). Key words: Micropropagation efficiency, exotic musa genotype, growth regulators.

High frequency multiple shoot induction from nodal segments and rhinacanthin production in the medicinal shrub Rhinacanthus nasutus (L.) Kurz

High frequency multiple shoots have been induced from nodal segments of Rhinacanthus nasutus (L.) Kurz., a potent anticancerous ethnomedicinal plant. For initiation of cultures, nodal segments were cultured on MS medium supplemented with various concentrations (1.0–5.0 μM) of 6-benzyladenine or thidiazuron (TDZ) alone or in combination with α-naphthalene acetic acid (NAA 0.5–1.0 μM). The optimum frequency of response (85 %) and shoot number (3.3) was observed on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The shoots developed on initiation media were excised and nodal segments were subcultured on MS medium supplemented with TDZ (4.0 μM) and NAA (0.5–1.0 μM). This subculturing process was repeated thrice, each with 45 days of duration and the multiple shoot formation was recorded at the end of every subculture stage. The highest frequency of response (100 %) and number of multiple shoots (24.1) per explant were recorded at the end of the third subculture passage on MS medium supplemented with 4.0 μM TDZ and 0.8 μM NAA. The optimum rooting of shoots was observed on ½ MS medium fortified with 3.0 μM indole-3-butyric acid. The rooted plants were successfully transplanted to soil. The estimation of rhinacanthin (RC) content in shoots and roots was carried out in 6-month-old ex vitro plants (i.e., plants regenerated via in vitro culture) and field grown natural plants by high performance liquid chromatography. Both shoots and roots of naturally grown plants showed slightly higher RC content than ex vitro grown plants. The highest RC content (4.6 mg/g DW RC-C, 0.14 mg/g DW RC-D and 0.10 mg/g DW RC-N) was recorded in roots of naturally grown plants.

Micropropagation of an exotic ornamental plant, Calathea crotalifera, for production of high quality plantlets.

A successful protocol was established for micropropagation in two selected varieties of exotic ornamental plants, Calathea crotalifera. The effects of different sterilization techniques, explant type, and the combination and concentration of plant growth regulators on shoots induction were studied. The axillary shoot buds explants sprouted from rhizomes in soil free conditions showed high induction rate of shoots with lowest contamination percentage when treated with combination of 30% (v/v) NaOCl, 70% (v/v) ethanol, and 0.3% (w/v) HgCl2. In the present study, the highest number of multiple shoots was obtained in MS basal medium supplemented with 3.5 mg/L 6-Benzylaminopurine (BAP), 1.0 mg/L 1-Naphthaleneacetic acid (NAA), 3% sucrose, and 6 g/L plant agar for both varieties and was used as multiplication medium. Microshoots were highly induced when the young shoot bud explants were incised longitudinally prior subculture. Chlorophyll analysis was studied to test the effects of activated charcoal and L-glutamine on reduction of necrosis problem. The maximum roots induction was recorded on MS medium supplemented with 1.0 mg/L 1-Naphthaleneacetic acid (NAA) compared to indolebutyric acid (IBA). The complete regenerated plantlets were successfully acclimatized in the soilless medium under greenhouse condition. This is the first report of rapid mass propagation for C. crotalifera.

High-frequency regeneration via multiple shoot induction of an elite recalcitrant cotton (Gossypium hirsutum L. cv Narashima) by using embryo apex

Cotton (Gossypium hirsutum L.) is one of the most commercially important fiber crops in the world. Compared with other crops, cotton represents a recalcitrant species for regeneration protocols. The development of efficient and rapid regeneration protocol for elite Indian cotton variety could help improve the quality characteristics and biotic or abiotic stress tolerance. Here we report a novel regeneration protocol in Indian cotton cultivar Narashima. The maximum number of multiple shoots obtained was 16 per explants, performance which has never been achieved in any prior reports. The embryo apex explants were isolated from 2 d old in vitro growing seedlings. Explants were cultured on MS medium containing different plant growth regulator combinations in order to induce multiple shoots. Among the tested combinations, the 2 mg/l of 6-benzylaminopurine (BAP) and 2 mg/l kinetin (KIN) proved to be most suited for achieving the maximum number of multiple shoots. The elongation of multiple shoots was obtained in media supplemented with gibberellic acid (GA3). The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. This method avoids callus tissue, the stage of regeneration which may lead to somaclonal variation. The important feature of the presented method is shortening of regeneration time, as well as the induction of a high number of multiple shoots per explants. The present protocol may provide an efficient and rapid regeneration tool for obtaining more stable transformants from embryo apex explants of Indian cotton cultivar Narashima.