Multiple Shoot Induction Research Articles

OPTIMIZATION OF GROWTH REGULATORS ON In vitro PROPAGATION OF Moringa oleifera Lam. AND PERFORMANCE EVALUATION OF FIELD GROWN TISSUE CULTURED PLANTS

Moringa oleifera is the most widely cultivated species of the Moringaceae family attributed to several medicinal uses and a high nutritional value. To obtain plantlets of drumstick tree (Moringa oleifera Lam.) with identical genetic make up for propagation or field production, in vitro cultures of drumstick tree were produced. For multiple shoot induction, the stem explants of drumstick tree plantlets were cultured on Murashige and Skoog (MS) agar medium supplemented with different concentrations of 6-benzyl adenine purine (BAP) and maintained at 25 ± 2°C for 5 weeks. The results showed that multiple shoot induction was present in the stem explants that were cultured on the MS medium supplemented with BAP at the concentration range of 0.5 - 3.0 mg/l or the combination of BAP at 1.5 mg/l, TDZ at the range of 0.2 - 1.0 mg/l and NAA at the range of 0.2 -1.0 mg/l. The MS medium containing BAP at 1.5 mg/l was found to produce 95.3% shoot formation with the highest average number of 8.4 shoots per explants. For plant regeneration, the shoot explants were cultured on ½ MS + 7 g/l agar + 15 g/l sucrose supplemented with IBA and IAA at different concentration root induction. The results showed that ½ MS + 7 g/l agar + 15 g/l sucrose supplemented with IBA at 0.3 mg/l and IAA at 0.2 mg/l produced 100% root formation with the highest average number of 4.2 roots per explants and 3.5 cm in length. The medium consisting of (40% soil + 50% sawdust + 10% worm compost) gave the highest average survival rate (88.9%) after 5 weeks under greenhouse conditions. Tissue culture protocol reported in this study is an alternative means of micro propagation of drumstick plantlets with uniform genotypes for breeding selection and field production.

Nutrition, age of explant and plant growth regulators affect the multiple shoot induction in Lantana camara L.

Nutrition, age of explant and plant growth regulators affect the multiple shoot induction in Lantana camara L.

Organogenesis on apical buds in common fig (Ficus carica) var. Black Jack

Plant tissue culture involves the use of explants obtained from plants to induce organogenesis with the help of plant growth regulators (PGRs). Micropropagation techniques provide a faster and economical solution to the limitations associated with traditional methods of plant cultivation. The present study focuses on the multiple shoot induction and proliferation of Ficus carica var. Black Jack. Factors that influence the growth of in vitro multiple shoots on the apical buds, which include growth media and PGRs, were investigated in this study. Different concentrations of cytokinins like 6-benzylaminopurine (BAP), Thidiazuron (TDZ), and Kinetin (Kin) were used on woody plant medium (WPM) for the optimization of media for multiple shoot induction and proliferation. Apical buds of Ficus carica var. Black Jack growing in WPM supplemented with BAP produced the healthiest plantlets, with the highest number of multiple shoots. The most efficient medium composition which produced the highest number of multiple shoots (37.8) per growing explant was WPM supplemented with 20 µM BAP. Proliferated multiple shoots were efficiently rooted using WPM + 20 µM BAP + 8 µM indole-3-acetic acid (IAA). This optimized medium composition significantly enhanced the production of multiple, disease-free plantlets using single apical bud explants of Ficus carica var. Black Jack. In the present study the observations indicate that WPM supplemented with 20 µM BAP is the best-suited medium for organogenesis and multiple shoot culture of Ficus carica var. Black Jack, and this technique can be potentially applied for commercialization of the plant. How to cite: Parab AR, Chew BL, Yeow LC, et al. Organogenesis on apical buds in common fig ( Ficus carica ) var. Black Jack. Electron J Biotechnol 2021;54. https://doi.org/10.1016/j.ejbt.2020.01.010

PLANT TISSUE CULTURE AND CALLUS INDUCTION FROM COTYLEDONARY SEGMENTS OF CUCURBITA MAXIMA (L)

The efficiency of regeneration from Cotyledonary segments was compared by containing the number of shoots MS basal medium with growth regulaters BAP 0.5mgl NAA and Kn, 0.5 mgl/L to 3.0 mgl/L, LGlutamic acid and CM Coconut milk 3.0mgl/L the regeneration MS contained medium 2.0 mgl/L BAP and 2.0 mgl/L L-Glutamic acid NAA Callus, shoot and plant regeneration in Cucurbita maxima the successful establishment of In Vitro requirements for plant regeneration from Cucurbita maxima Cotyledonary nodal segments. The nodal explants were inoculated on MS basal medium supplemented with various cytokinins i.e., BAP and NAA. Coconut water also had a role in triggering the formation of multiple shoots. Addition of BAP at 2.0 mg/l concentration or NAA at 3.0 mg/l to the MS basal medium, induced regeneration from the nodal segments Trichosanthes Cucumeriana. In Vitro seeding derived callus saussurea (Joshi 2003, Wawro Sch 2001). Elimination of viruses fro fruit trees (Knapp et al 1995, Cieslinska 2002) Shoot induction was achieved in one of the important medicinal plant of cucurbitacae family, Cucurbita maxima. MS medium supplemented with 1.0 mg/l BAP + 2.0 mg/l NAA and 2.0 mg/l L-Glutamicacid was found to be optimum to induce shoots. The present study established reliable and reproducible protocol for rapid multiple shoot induction from Cotyledonary explants of Trichosanthes Cucumeriana using different concentrations and combinations of cytokinins. Tissue culture techniques are now becoming popular as alternative means of vegetative propagation. The effect of benzyl amino purine in inducing shoot induction was already reported in some of the important medicinal plants (Komalavalli and Rao, 2000). An effective and reproductive procedure for the regeneration of shoots and plantlets from callus and in vitro tissue culture technique is essential in studies involving gene transfer. Somewhat greater success in regeneration of bean from organ cultures Malik and Sexena (1991) protoplast Isolation from leaf explants of Solanum Surattense A Ramulu, et al (2014) Phytochemical analysis and biological activates in Solanum Surattense Venakteshwarlu et al (2018)

Analysis of correlation between seed vigour, germination and multiple shoot induction in soybean (Glycine max L. Merr.)

The physical and physiological roles of seed properties are often neglected during plant tissue culture. These properties determine the level of activity and performance of seeds, which is commonly known as seed vigour. This paper reports on the role of seed vigour on seed germination and shoot induction using cotyledonary node explants. For this, explants prepared from soybean seedlings established using seeds stored under ambient conditions for different durations (0, 3, 6 and 9-months) were used. The findings indicated that seed germination was highly instantaneous after harvest and began to decrease as seed storage was prolonged for 3, 6 and 9-months, respectively. Similar observations were made during shoot induction. Generally, the analysis revealed a positive relationship between seed vigour, germination and multiple shoot initiation as indicated by the Pearson's correlation coefficient reported. According to the findings, seed vigour could serve as a major obstacle to efficient germination and shoot proliferation for subsequent in vitro plant regeneration.

Multiple shoot induction in zygotic embryos: a strategy for acceleration of banana breeding

The presence of residual female fertility in most of the parthenocarpic banana accessions encourages the banana breeder to develop new hybrids through conventional breeding. Desirable trait can be fixed in the first generation of hybrid progenies, but the evaluation of these hybrids in field is the time-consuming process owing to non-availability of uniform suckers/planting material. This can be overcome by developing multiple shoots from single embryo in a short period of time through embryo culture. A protocol for in vitro multiplication of plantlets from zygotic embryos was standardized in seeded accessions. Multiple shoots from zygotic embryos were achieved up to 55.2% and 64.1% in seeded accessions of Musa acuminata and M. velutina respectively in medium supplemented with 17.76 µM of BAP. The Single shoot derived (only germination) from zygotic embryos was decapitated and the apical meristem were disturbed for further multiple shoot formation in media supplemented with 17.76 µM of BAP. Present studies revealed that in total 75% and 91% of the M. acuminata and M.velutina embryos were able to produce multiple shoot from single embryo by manipulating the media composition and decortications technique. The above protocol was applied for zygotic embryos obtained from controlled pollination (18 cross combinations) and open pollination (nine accessions) of various genomic groups (ABB, AAB, AA). The multiple shoots derived from zygotic embryos and plantlet germinated from zygotic embryos was examined for genetic fidelity analysis by SSR markers.

Regeneration and micropropagation of Meyer lemon (Citrus x meyeri) supported by polymorphism analysis via molecular markers

Meyer lemon (Citrus x meyeri) is a valuable sweet hybrid Citrus from China, renowned for its unique taste and high nutritional value. The current study aims to establish an efficient micropropagation protocol and to evaluate the potential regenerated somatic embryos induced from in vitro Meyer lemon explants. Shoot tips and epicotyl segments were inoculated in half strength Murashige and Skoog (½ MS) medium supplemented with plant growth regulators (PGRs) for multiple shoot and root induction prior to polymorphism and microscopy analysis. Benzylaminopurine (BAP) treatment at 1 mg L−1 was found optimal in the induction of multiple shoots (100% induction rate) with the average of 3.29 ± 0.2 shoots. The synergistic effects of 2 mg L−1 BAP and 1.5 mg L−1 Indole-3-acetic acid (IAA) resulted in maximum shoot regeneration frequency (100%) with an average number of 3.6 ± 0.5 shoots per explant. Woody plant medium (WPM) without PGRs exhibited the highest rooting frequency (100%) and acclimatization of in vitro plantlets yielded 82% survivability. Inter simple sequence repeat (ISSR) and start codon targeted (SCoT) markers revealed no somaclonal variation occurrences between micropropagated plantlets of five subculture cycles and mother plant. Potential indirect somatic embryogenesis was observed from epicotyl segments of in vitro germinated seedlings at an average of 7.3 ± 1.0 embryos per explant with 80% response in ½ MS with 0.5 mg L−1 thidiazuron (TDZ). Morpho-histological and scanning electron microscopy (SEM) observations confirmed the initiation and developmental features of the embryos. This study has successfully established an efficient micropropagation protocol for Meyer lemon and documented the potential initiation of indirect somatic embryogenesis.

Cytotoxic, clonal fidelity, and antioxidant evaluation of in vitro propagated Vitex negundo var. cannabifolia

Vitex negundo L. var. cannabifolia (Sieb. et Zucc.) Hand.-Mazz. is an important medicinal plant rich in bioactive compounds. In vitro tissue culture can be used to produce high-quality starting materials for commercial cultivation. However, the micropropagation technique, especially the comprehensive evaluation of regenerants at the metabolite level, is not well known in this plant species. Employing dormant buds as explants, we developed direct and callus mediated indirect propagation protocols for this species and then evaluated the antioxidant and cytotoxic activities of regenerants. The highest multiple shoot induction frequency of 100%, with a proliferation coefficient of 20.3, was achieved using Murashige and Skoog basal medium with 4.4 µM 6-benzylaminopurine and 1.1 µM 1-naphthylacetic acid. The same medium supplemented with 31.5 µM copper sulfate was optimal for callus differentiation, with a shoot regeneration rate of 81.10%. Inter-simple sequence repeat analysis showed a high genomic fidelity of in vitro regenerants. Chemical, physiological, and cytotoxic analyses indicated that in vitro propagated plants at the adult stage are indistinguishable from wild-grown plants. In particular, the cytotoxicity of V. negundo var. cannabifolia to breast cancer cell line SUM159 reveals the potential of this species for use in treating human breast cancer, with cytotoxic compounds beyond vitexin also apparently observed. Our results indicate that V. negundo var. cannabifolia can be reliably micropropagated in vitro. Moreover, the regenerants have high cytotoxic activity against highly metastatic human breast cancer. We developed efficient micropropagation protocols for Vitex negundo var. cannabifolia, and found the regenerants were consistent with wild-grown plants according to the assessments at the genomic, metabolic, physiological, and cytotoxic levels. We also found the high anticancer activity of V. negundo var. cannabifolia, and confirmed that there are other cytotoxic compounds other than vitexin.

In vitro propagation and cytological analysis of Sophora mollis Royle: an endangered medicinal shrub

BackgroundSophora mollis Royle (family Fabaceae, subfamily-Papilionaceae) is a multipurpose legume distributed in plains and foothills of the North-West Himalaya to Nepal and is facing high risk of extinction due to habitat loss and exploitation by the local people for its fuel and fodder values. Therefore, the present study was conducted to standardize a micropropagation protocol for Sophora mollis by using shoot tip explants and to study the meiotic chromosome count in the species. ResultsMultiple shoots were induced in shoot tip explants of Sophora mollis in Murashige and Skoog medium supplemented with different concentrations of cytokinins alone (BAP, TDZ, and Kinetin) and in combination with varying concentrations of NAA. MS medium supplemented with BAP (8.9 μM) was observed to be the optimal medium for multiple shoot induction and maximum 25.32 shoots per explant was obtained with average length of 4.5 ± 0.8 cm. In vitro developed shoots were transferred onto rooting media supplemented with different concentrations of auxin (IAA, IBA, and NAA). Maximum 86% rooting was observed in half-strength MS medium supplemented with 21.20 μM NAA with an average of 21.26 roots per culture. In vitro raised plantlets were adapted to greenhouse for better acclimatization and 60% plants were successfully transferred to the open environment. Based on the chromosome counts available from the literature and the current study, the species tend to show a basic chromosome number of x = 9. ConclusionThe micropropagation protocol standardized can be helpful for the ex situ mass multiplication and germplasm conservation of the endangered species. Moreover, the ex situ conservation approach will be helpful in actively bridging the gap between ex situ and in situ approaches through the reintroduction of species in the wild. The cytological studies revealed the basic chromosome number x = 9 of the species.

Effects of Benzyladenine on in vitro ‘Hom Rangsi’ Rice and Induction of Aluminum Acid Tolerance Lines by Gamma Irradiation

‘Hom Rangsi’ was the non-photoperiod aromatic mutant rice which derived from fast neutron radiation KDML 105. ‘Hom Rangsi’ seeds were cultured on MS solid medium without any supplemented for a week. And then, all explants were placed on MS (Murashige and Skoog, 1962) medium supplemented with 0, 5, 10, 15, 20, 25 mg/L BA (benzyladenine) for multiple shoot induction. The optimal concentration of BA for induced multiple shoot induction of ‘Hom Rangsi’ line was MS + BA 25 mg/L, the highest number of shoots were 5.38 shoot/seed. The following experiment was done, irradiated ‘Hom Rangsi’ seeds with 0, 100, 200, 300, 400 Gy gamma ray which cultured on MS solid medium supplemented with 400 mg/L Al3+ pH 2.9 were selected for acid tolerance lines. After six weeks cultured, the survivals of irradiated plantlets were 86.32, 77.78, 58.95, 58.95, 21.87% and the height of irradiated plantlets were 8.4, 8.3, 6.7, 6.6, 6.1 cm respectively without any shoot budding. All survival plantlets were transferred to suitable MS + BA 25 mg/L medium which discovered from the first experiment for multiple shoot budding. After six weeks cultured, the maximum of 5.24 shoots/plantlet were found from 300 Gy irradiation significantly and followed by 400, 200, 0 and 100 Gy irradiation treatments which gave 4.55 and 4.41, 4.37 and 4.31 shoots/planlet respectively.

Multiple shoot induction and plant regeneration of Staurogyne repens (Nees) Kuntze

Multiple shoot induction and plant regeneration of Staurogyne repens (Nees) Kuntze

High Efficiency In vitro Regeneration and Genetic Stability of Corallocarpus epigaeus - An Endangered Medicinal Plant

Indirect regeneration of plantlets from multiple shoot induction of Corallocarpus epigaeus was obtained from leaf and nodal explants on MS with different concentrations of BAP in combination with IAA/IBA or IBA alone. Among all the combinations, BAP and IBA exhibited maximum regeneration. High frequency of multiple shoots (89%) was obtained on BAP (2.0 mg/l) and IBA (1.5 mg/l) in nodal explants. Maximum mean shoot length of 6.8 ± 0.33 cm was obtained in nodal explants cultured on BAP (1.0 mg/l) + IBA (0.5 mg/l), followed by leaf explants with 6.7 ± 0.47 cm on BAP (3.0 mg/l) + IAA (2.5 mg/l). The highest frequency of rooting (88.3%) was obtained on NAA (1.0 mg/l) and IBA (2.0 mg/l) with 21.83 ± 0.57 mean number of roots. The well-rooted healthy plantlets were acclimatized with a survival rate of 80%. Inter simple sequence repeat (ISSR) analysis revealed the genetic similarity of in vitro raised plants with the mother plant.
 Plant Tissue Cult. & Biotech. 30(2): 219-229, 2020 (December)

Enhancement of adenine sulfate and growth promoting substances on multiple shoot induction of Sangmon ‘Nuan Rajinee’ bamboo (Dendrocalamus sericeus Munro)

ISHS I International Symposium on Botanical Gardens and Landscapes Enhancement of adenine sulfate and growth promoting substances on multiple shoot induction of Sangmon 'Nuan Rajinee' bamboo (Dendrocalamus sericeus Munro)

Highly efficient rapid micropropagation and assessment of genetic fidelity of regenerants by ISSR and SCoT markers of Solanum khasianum Clarke

An efficient method of rapid micropropagation of Solanum khasianum Clarke was successfully established from the leaf, petiole, and nodal explants. The morphogenetic response of different concentrations of TDZ and BAP individually or in combination with auxins (IAA/IBA/2,4-D) was tested. Friable callus was obtained on different concentrations of BAP alone or in combination with IAA/IBA/2,4-D. Rapid multiple shoot induction was achieved from friable callus on MS medium supplemented with varying concentrations of TDZ and IBA. The leaf explants exhibited a high frequency of multiple shoots than petiole and nodal explants with an optimal percentage of response (92.73%), mean shoot number (53.5 ± 0.47), and shoot length (11.2 ± 0.53 cm) on MS medium augmented with TDZ (1.5 mg l−1) and IBA (1.5 mg l−1). Maximum rooting efficiency was achieved on MS medium with 1.5 mg l−1 IBA with 12.8 ± 0.36 mean number of roots. The in vitro rooted plants were acclimatized with a survival rate of 80%. The genetic fidelity of the regenerants assayed by the ISSR and the SCoT markers showed no genetic variation. The study examined the micropropagation responses of S. khasianum in the presence of various growth regulators and provided a simple and more suitable protocol adapted for the mass propagation of clones in this species. We have established a highly efficient micropropagation system for large scale production in Solanum khasianum. Evaluation of clonal fidelity by using ISSR and SCoT markers detected no somaclonal variations. The present study helps to the enhancement of potential alkaloids (solasodine) with the help of biotechnological tools.

Optimization of growth regulators on in vitro propagation of Moringa stenopetala from shoot explants

BackgroundMoringa stenopetala belongs to the flowering family Moringaceae and genus Moringa. It is often referred to as the East African Moringa tree because it is native only to southern Ethiopia and northern Kenya. The expansion of its cultivation and utilization throughout the world especially in Africa is becoming important. For such expansion, the existing propagation method is limiting, so it needs a good propagation system to supply enough planting material with a uniform genotype. Therefore, the main objective of this study was to optimize an in vitro shoot multiplication protocol for M. stenopetala by using shoot tip as explants.ResultsShoots were sterilized and cultured on Muraghige and Skoog (MS) medium for in vitro shoot initiation. For multiple shoot induction, the explants were cultured on MS medium supplemented with different concentrations of kinetin (0.5, 1.0, 1.5, 2.0, 2.5 mg/L) with Indole-3- butyric acid (IBA) or α -naphthalene acetic acid (NAA) (0.01, 0.1, 0.5 mg/L) and maintained at 25 ± 2 °C for four weeks. Rooting was achieved by culturing well developed shoots in half-strength MS medium containing IBA (0.1, 0.5, 1.0, 1.5, 2.0 mg/L), NAA (0.1, 0.5, 1.0, 1.5, 2.0 mg/L), and 0.5 mg/L IBA with NAA (0.1, 0.5, 1.0, 1.5, 2.0 mg/L). Statistical analysis revealed that there was a significant difference among all treatments applied in both shoot multiplication and rooting experiments. The maximum number of shoots per explant (3.43 ± 1.41) and 7.97 ± 4.18 leaves per explant were obtained on MS medium containing 0.5 mg/L kinetin with 0.01 mg/LNAA. The highest mean number of roots per shoot (1.63 ± 1.03) and mean root length (0.87 ± 1.22 cm) were obtained on MS medium containing 1.0 mg/LNAA and 0.1 mg/LIBA alone respectively. After acclimatization, 76% of plants were survived in the greenhouse.ConclusionIn general, using NAA with kinetin for shoot multiplication was effective than kinetin with IBA. On the other hand, the application of 1.0 mg/L NAA alone and 1.0 mg/L NAA with 0.5 mg/L IBA were more effective for root induction.

Citrus TISSUE CULTURE WITH TWO DIFFERENT APPROACHES

Citrus is an important genius for economy and human health but a susceptible genius against biotic and abiotic stresses, so it needs to improved programs. Different basal media (MS, ½MA, ?MS and DKW) and different kind and concentrations of plant growth regulators i.e. BA, KIN, 2ip, ZE and TDZ (0 – 2 mg/l) and NAA, IAA and IBA (0 – 2 mg/l) added with 30 g/l sucrose, 3 g/l active charcoal and 7.5 g/l bacteriological agar] were used for organogenesis include shooting and rooting, also callusing from nodal explant of Citrus latifolia. MS medium supplemented with 1 mg/l BA, and 0.01 mg/l NAA is the best media for multiple shoot induction on nodal explants and elongation of them. Other cytokinins had not significant effects on shoot induction and multiplication. Using of 0.01 mg/l IBA instead of 0.01 mg/l NAA on medium with 1 mg/l BA, led to multiple shoot induction on nodal explant indirectly. Rooting was induced on DKW medium plus 1.5 mg/l NAA in the best way compared another media. Both direct and indirect organogenesis (multiple shoot induction) were carried out on media with very similar contents. So we can use very simple and practical methods tissue culture for different improved programs in Citrus genius genius.

Complete plant regeneration of Valeriana wallichii DC. on auxin enriched medium and phytochemical analysis

Valeriana wallichii DC. (Valerianaceae) is a well-known medicinal herb distributed in Northwest Himalayas. The herb is utilized in the treatment of numerous ailments and diseases such as diarrhoea, diabeties, gastrospans, ulcer and wound healing, etc. Overexploitation, especially collection of rhizomes on a large scale for the medicinal purpose has significantly declined the availability of the herb in natural stands. Hence, there is a requirement of development of cultivation practices and protocol for mass propagation to achieve sustained utilization along with conservation of the species. In the present study, in-vitro culture of nodal segments on MS+2iP+IAA+2,4-D and MS+2iP+IAA+NAA resulted in multiple shoot induction along with regeneration of in-vitro roots on the same medium making the protocol cost-effective, efficient and comparatively less time-consuming. Moreover, the regeneration of adventitious roots from regenerated shoots enhanced the total number of plants obtained per explant as shoots with adventitious roots were individually excised and were transferred to natural conditions through the process of acclimatization. GC-MS analysis of a methanolic extract of leaves of the mother plant and micropropagated plant revealed the presence of 37 and 36 phytocompounds respectively. Phytocompounds including eucalyptol, neophytidiene, hexadecenoic acid, dimethyl palmitamine were identified to be present in the leaves of both mother and micropropagated plants whereas other compounds such as eicosyne, lilial, behenic alcohol were confined to be present in extract prepared from leaves of mother plant. Similary some phytocompounds (phytol, vinicizer, retinol) were detected in methanolic extract prepared from leaves of micropropagated plants.

IMPROVEMENT OF THE PROCESSES OF MICROCLONAL REPRODUCTION OF BLACKBERRY RUBUS CAESIUS L. VAR. THORNFREE

Aim. The initial stages’ improvement of microclonal propagation of Blackberry Rubus caesius L. var. Thornfree, particularly the introduction to in vitro culture for subsequent effective cultivation. Materials and methods. Methods of introduction of initial explants of blackberry to in vitro culture and methods of microclonal reproduction were used. Two schemes of sterilization of plant material of Blackberry with the use of fungicidal drugs Hinozol and Horus were tested to identify the most optimal for this type of plants. The effect of agar concentration in the medium, the consistency of the nutrient medium on the processes of survivability, bud proliferation and induction of multiple shoots of Blackberry was studied, and semi-liquid medium was used for the first time. The medium was prepared semi-liquid (4 g/l of agar) and solid (8 g/l of agar). Results. The optimal sterilization scheme of plant material for the introduction of Blackberry to in vitro culture was determined and the most effective fungicidal drug – Horus was selected. It was found that the use of semi-liquid Murashige and Skoog nutrient medium for the initial stages of microclonal propagation of Blackberry to in vitro culture allows: to increase the survival of explants by 30%compared to solid medium; to accelerate the proliferation of axillary buds for 1–2 days; to increase the intensity of the additional shoots formation of Blackberry in vitro in 6–7 times. Conclusion. During the initial stages of microclonal propagation of Blackberry Rubus caesius L. var. Thornfree, it is advisable to use semi-liquid nutrient medium to improve the survival of explants, faster proliferation of buds and the formation of new multiple shoots in vitro.

Mutagen treatment affects the regeneration efficiency of the shoot-tip explant in Celosia cristate L. (Cockscomb)

The shoot-tip explant harvested from ethyl methane sulphonate (EMS) and gamma ray (GR) mutagenized seedling was cultured over MS medium fortified with NAA and BAP for five generations to amplify the mutated sector. Mutagens reduced the regeneration efficiency of the explant and affected its plant growth regulator-dependence for multiple shoot induction. While the 12d-old shoot-tip from GR-treated seedling induced shoots with 0.5µM NAA+6.6µM BAP; that from EMS-treated seedling induced shoots with 8.8µM BAP. The present study establishes that the mutagens affect the regeneration process in the explant.

Direct shoot organogenesis from shoot tip explants of a highly medicinal valued tree Pterocarpus marsupium Roxb.

An in vitro regeneration system for propagation of a valuable medicinal tree, Pterocarpus marsupium, using shoot tip (ST) explants derived from 7-d-old axenic seedlings has been successfully developed. Murashige and Skoog (MS) medium containing cytokinins (BA, mT, or Kn) and auxins (NAA, IAA) in different concentrations and combinations showed to have a marked stimulatory effect on the regeneration output. Of cytokinins, meta-topolin (mT 7.0 μM) proved optimum dose for multiple shoot induction yielding 6.50 ± 0.49 shoots per explant with mean shoot length (4.00 ± 0.16 cm) in 70% cultures after 6 wk. Whereas, supplementation of low concentration of auxin (1.0 μM NAA) with optimal cytokinin (7.0 μM mT) favored enhanced shoot induction with increased percent response. At this level, a maximum of 13.54 ± 0.34 shoots per explant with (5.30 ± 0.10 cm) mean shoot length were recorded in 80% cultures after 12 wk. Thereafter, microshoots were isolated and their basal end treated with high doses of indole-3-butyric acid (250 μM) for 5 d followed to transplant onto various potting substrates for ex vitro root formation. A maximum of 3.63 ± 0.0.08 roots per microshoot and mean root length of 3.59 ± 0.07 cm with highest rooting frequency (67.7%) was observed after 4 wk of transplantation. During ex vitro rooting-cum-acclimatization, various physiological parameters were found to be fluctuating initially for 35 d; thereafter, an increasing trend was observed in net photosynthetic rate, intercellular CO2 concentration, and chlorophyll a/b estimation. Overall, the successfully acclimatized regenerants when transferred in natural condition showed about 96.7% survival rate after 3 mo. Assessment of genetic integrity of tissue culture raised plantlets was accomplished by DNA–based ISSR primers. A total of 35 bands with an average of 4.38 bands per primer were scored in monomorphic banding pattern. Thus, it is apparent that the mode of in vitro regeneration protocol was suitable for obtaining true-to-type and disease-free plants.