Rooting Of Microshoots Research Articles

MICROPROPAGATION FOR THE CONSERVATION OF RARE AND ENDANGERED MALUS NIEDZWETZKYANA

The conservation of biological diversity is one of the most important tasks at present given the strong association of biodiversity with food security and improving nutrition. Currently, 387 plant species are listed in the Red Book of Kazakhstan, including the rare, endemic, and endangered species Malus niedzwetzkyana, which is also included on the International Red List. Biotechnology methods are currently widely applied to preserve such rare plant species, which allows for the long-term preservation of genetic material, enabling their large-scale reproduction and propagation. One of the most effective methods for this purpose is microclonal propagation, which is similar to the vegetative method of reproduction except that the entire process proceeds with an in vitro culture system. The resulting clones are healthy and genetically stable. In this study, we optimized the microclonal propagation technique for Malus niedzwetzkyana. Axillary buds of annual shoots were used as the initial material. The mode of sterilization of the axillary buds, composition of the nutrient medium for establishment of in vitro culture, and the multiplication and rooting of microshoots were optimized. The data obtained provide an essential resource for sustaining the reproduction and propagation of this rare and endangered species of apple trees.

Factors affecting the efficiency of in vitro regeneration from seedling-derived nodal explants of Nyctanthes arbor-tristis L. and evaluation of genetic fidelity

An in vitro micropropagation protocol for Nyctanthes arbor-tristis was developed through the culture of seedling-derived nodal explants. The highest direct shoot induction rate, the mean number of shoots per explant and average shoot length was obtained on MS medium supplemented with 5 mg/l 6-benzylaminopurine (BAP). Full-strength MS medium was found better than other concentrations for organogenesis. Their lower or higher concentration from optimum level was inhibitory for shoot regeneration. Among different carbon sources tested for organogenesis, MS medium supplemented with 87.64 mM sucrose was superior to other carbon sources for in vitro morphogenesis. Best rooting of microshoots was achieved on one-quarter strength MS medium supplemented with 2 mg/l indole-3-butyric acid (IBA). Under this treatment, maximum mean number of roots/shoot and mean root length were 9.40 ± 0.70 and 2.47 ± 0.25 cm, respectively. The genetic fidelity among regenerates and donor plant has been confirmed by ISSR markers.

In vitro propagation, clonal fidelity and phytochemical analysis of Rhodiola imbricata Edgew: a rare trans-Himalayan medicinal plant

The present study focuses on development of a micropropagation protocol for true to type plants of Rhodiola imbricata, an endangered medicinal plant found in trans-Himalayan Leh-Ladakh region of India. It also aims at analyzing the pharmaceutically important secondary metabolites and antioxidant potential of in vitro and in vivo plants. Various cytokinins and auxins were tested for shoot proliferation and in vitro rooting of the microshoots, respectively. Random primers were used for checking genetic uniformity at different stages of micropropagation. Pharmaceutically important secondary metabolites of R. imbricata such as Rosavin, total polyphenols and free radical scavenging activity were analyzed by HPLC. Among different cytokinins used, BAP (5 µM) and TDZ (1 µM) were found to perform better in terms of shoot proliferation, shoot length and number of leaves as compared to other concentrations. For rooting of microshoots, a lower concentration of NAA (0.5 µM) yielded more efficient rooting of micro shoots (17.33 roots per micro shoot). In vitro rooted microshoots were hardened and showed 60% survival rate. The content of gallic acid, chlorogenic acid and 4-hydroxybenzoic acid was higher in the in vivo plant. The amount of ferulic acid was higher in the in vitro raised plant when compared to field grown plant. Furthermore, caffeic acid and p-coumaric acid were higher in the in vitro raised plants as compared to field grown plants. This work will facilitate in conservation of this endangered herb and provide necessary plant materials for various biotechnological and pharmaceutical applications.

Direct Shoot Organogenesis from Seedling Derived Shoot Tip Explants of Endangered Medicinal Plant Saussurea costus (Falc.) Lipsch.

An efficient direct in vitro plant regeneration protocol for Saussurea costus (kuth) was developed using shoot tip explants derived from in vitro grown seedlings to generate genetically uniform plants. Murashige and Skoog (MS) medium supplemented with 1.14 µM thiadiazuron and 2.68 µM naphthalene acetic acid was found to be most optimal for highest for average shoot regeneration (73.33%) with maximum average number of shoots (11.4) and average shoot length (4.17 cm). For in vitro multiplication, shoots cultured on MS medium supplemented with 4.44 µM benzyl adenine, 2.32 µM kinetin and 1.44 µM gibberellic acid were found to grow best with 20.7 average shoot multiplication rate after 5–6 weeks. MS medium supplemented with 2.46 µM indole butyric acid was found to be the best medium for rooting of microshoots (77.78% rooting with 6.0 average number of roots and 3.07 cm average root length). The in vitro raised plantlets were successfully acclimatized under ex vitro conditions. The true to type nature of the in vitro raised plants was confirmed using DNA based ISSR markers for assessment of genetic stability of micropropagated plants. The standardized micropropagation protocol under present study may be tuned towards mass production of roots which are the most economical part of the plant. This would be useful as prerequisite for carrying out any genetic transformation studies while concentrating on any desirable trait in the same herb.

Development of in vitro protocol for mass multiplication of open pollinated seedling of rose (Rosa × hybrida L.) cv. Rose Sherbet

An in-vitro protocol for promising open pollinated seedling of rose (Rosa × hybrida L.) cv. Rose Sherbet was developed for mass multiplication. Murashige and Skoog (MS) medium supplemented with 5.0 mg/l BAP + 0.1 mg/l NAA+ 0.5 mg/l GA3 was found most effective for culture establishment. The maximum number of micro shoots (36.67 shoots/explant) were induced on MS medium comprising 5.0 mg/l BAP + 0.1 mg/l NAA+ 0.5 mg/l GA3 along with 40 mg/l adenine sulphate and was found to be better for shoot proliferation. The highest shoot length after 15 days (1.90 cm), 30 days (3.25 cm) and 45 days (4.65 cm) was observed on MS medium supplemented with 1.0 mg/l GA3. Rooting of micro shoots was successfully induced on half strength MS basal medium supplemented with NAA (0.5 mg/l) rooting growth regulators. The regenerated plantlets were efficiently hardened in glass jars filled with coco peat + vermiculite + perlite (1: 1: 1) moistened with half strength MS medium salts and covered with polypropylene lids, thereafter plants were successfully transferred to the field with good survival.

Влияние питательной среды и спектрального состава света на размножение земляники in vitro

The research was conducted in 2016-2017 on the basis of a meristem laboratory. Object of the research is the micro-shoots of garden strawberry the Festival variety and remontant strawberry the Brighton variety at the stages of micropropagation and rooting. The following nutrient media were studied: Murashige and Skoog (control), Murashige and Skoog modified (microelements were replaced with a corresponding dose of microfertilizer “Siliplant”) and Boxyu; LED irradiators - with a ratio in the spectrum of red, blue and white light R2: B1:W1 and R1: B1: W1, respectively, and a programmable one with a changing spectrum. A luminescent lamp with white light served as the control. The highest rate of reproduction in the cultivation of garden strawberry in vitro was obtained using the Murashige and Skoog culture medium under a LED irradiator with a R1: B1: W1 combination of red, blue and white light and the irradiator with a changing spectral composition: 6.0 and 6.2 pieces/microcutting, respectively. The highest reproduction factor of remontant strawberry was provided by cultivation on the Murashige and Skoog nutrient medium using the LED light irradiator in ratio of red, blue and white as R1:B1:W1 and the irradiator with a changing spectral composition: 3.5 and 3.2 pieces / microcutting, respectively. A rooting of micro-shoots of garden strawberry on the modified Murashige and Skoog nutrient medium and Boxyu provided a significant increase in rooting ability to 100% and 97.5%, respectively. Cultivation on the Boxyu nutrient medium has increased the rooting ability of micro-shoots of remontant strawberry to 95.0%. All the LED irradiators provided a significant increase in the rooting ability of micro-shoots: for garden strawberries - up to 96.6-100%, for remontant - up to 83.3-90.0%.

Ex vitro rescue, physiochemical evaluation, secondary metabolite production and assessment of genetic stability using DNA based molecular markers in regenerated plants of Decalepis salicifolia (Bedd. ex Hook.f.) Venter

To ensure replenishment, a refine protocol for micropropagation of Decalepis salicifolia (Bedd. ex Hook.f.) Venter a critically endangered and endemic medicinal plant was developed using mature nodal explants. A high frequency shoot regeneration system was obtained on Murashige and Skoog (MS) medium comprised of 6- benzyladenine (BA) (5.0 µM) + α-naphthalene acetic acid (NAA) (0.5 µM) + adenine sulphate (ADS) (30.0 µM) corresponds to a highest mean number of 9.97 ± 0.01 shoots/explants with maximum shoot length of 6.46 ± 0.1 cm. Successful rooting in microshoots was achieved on half strength MS medium supplemented with indole-3-butyric acid (IBA) (2.5 µM). A maximum of 6.10 ± 0.07 roots/microshoot with average root length of 2.30 ± 0.06 cm was obtained. As much as 90% plantlets survived when Soilrite™ was used as planting substrate and finally established in soil without any casualty and morphological variation. Acclimatized plantlets were screened for pigment content, net photosynthetic rate (PN), stomatal conductance (Gs) and transpiration rate (E) during subsequent days of acclimatization as well as the changes in antioxidant was also evaluated. A steady rise was observed in the activity of superoxide dismutase (SOD) for initial 21 days and then after a decrease was found showing improved acclimatization efficiency of the plant. Similarly, the activities of catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) enzyme shows reliable increase as the days of acclimatization advanced which play their precautionary role against oxidative damage to the plant. The genetic fidelity of the in vitro raised plantlets with that of mother plant was further confirmed using random amplified polymorphic DNA (RAPD) and inters simple sequence repeats (ISSR) analysis. Additionally, the effect of acclimatization on the biosynthesis of 2-hydroxy-4-methoxybenzaldehyde (2H4MB) in the root system was also evaluated in relation to their biomass production. Maximum fresh weight (4.9 g/plant), dry weight (0.65 g/plant) of roots and 2H4MB content (6.8 µg ml−1 of root extract) was obtained after 10 weeks of acclimatization. Accelerated multiplication rate with the stability of genetic virtue, physiological and biochemical parameter assure the efficacy of the protocol developed for the propagation of this critically endangered medicinal plant.

Establishment of in vitro propagation protocol for Hybrid Tea rose cv. Raktagandha

An efficient protocol for in vitro multiplication of hybrid tea rose cv. Raktagandha was developed using axillary bud segments. Out of different pre-treatments of explants tried, the highest explant survival (78.05%) was obtained with carbendazim (0.2%) + diathane M-45 (0.2%) + 8-HQC (200 mg/l) for 3 h on a horizontal shaker (120 rpm). Sucrose concentration of 30 g/l in the medium seems to be optimal for in vitro shoot multiplication. Murashige and Skoog medium supplemented with 3.5 mg/l BAP + 0.2 mg/l NAA + 0.5 mg/l GA3 was found most effective for culture establishment and shoot proliferation with highest number of micro-shoots (4.62 shoots/explant). Rooting of micro-shoots was induced on half-strength MS basal medium supplemented with NAA (0.5 mg/l) + IBA (0.5 mg/l). The regenerated plantlets were efficiently hardened in glass jars filled with vermiculite + agropeat (1:2) moistened with half-strength MS medium salts and covered with polypropylene lids, thereafter plants were successfully transferred to the glasshouse with good survival.

In vitro proliferation and ex vitro rooting of microshoots of commercially important rabbiteye blueberry (Vaccinium ashei Reade) using spectral lights

Efficient protocols for in vitro shoot growth and ex vitro root formation from in vitro-derived shoots under light-emitting diodes (LEDs) with simple ventilation were developed for rabbiteye blueberry cv. ‘Titan’. Red LEDs promoted shoot elongation while blue LEDs induced short shoots with enhanced accumulation of leaf chlorophyll contents. A combined treatment of 80% red with 20% blue LEDs was found the most suitable for plant growth, and more effective than 50% red mixed with 50% blue LEDs or fluorescent lamps as the control treatment. The use of ventilated vessels with ambient CO2 enrichment from a growth chamber resulted in healthy plants having greatly improved shoot length, leaf expansion, leaf chlorophyll contents and dry weight of both shoots and roots. In vitro-cultured shoots could be directly rooted at 100% under non-sterile conditions using a perlite-peat mixture with careful controls of high humidity. These results suggest that LEDs, particularly when coupled with ventilated vessels, should be used as a primary light source for replacing conventional fluorescent tubes in large-scale production of rabbiteye blueberries.

Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin

Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77 × 105 protoplasts (Pp) per gram fresh weight with 94 % viability; after 60 min pre-plasmolysis with 0.7 M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7 M mannitol, 1.5 % cellulase Onozuka R10, and 1 % pectolyase Y-23 for 6 h. Liquid Kao and Michayluk medium containing 2.7 μM α-naphthaleneacetic acid (NAA) and 2.2 μM 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3–5 × 105 Pp ml−1. Protoplast-derived microcalli became visible after 3–4 weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4 μM BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2 μM BA plus 4.6 μM zeatin after 2–3 weeks, with an overall shoot organogenesis efficiency of 78–93 %. Rooting of microshoots on half-strength MS medium containing 4.9 µM indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of >62 %. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings.

In-vitro protocol for mass multiplication in bougainvillea (Bougainvillea sp) cv. Mahatma Gandhi and Refulgens

Bougainvillea (Bougainvillea sp) is recognized as a high value landscape plant. It is usually propagated by hardwood cuttings, but in certain varieties, rooting per cent is very low, ultimately the rate of multiplication is less. Therefore, the present investigation was carried with an objective to standardize the protocol for quick, easy and mass multiplication of two difficult-to-root cultivars Mahatma Gandhi and Refulgens, using in vitro techniques. Nodal sections with axillary buds were excised, surface-sterilized and cultured on MS medium supplemented with plant growth regulators. Pre-treatment agitation of explants in carbendazim (0.1%) + Metalaxyl (0.1%) + 8-HQC (200 mg/l) for 3 hr followed by quick dip in ethyl alcohol (70%; v/v) for 30 sec and surface sterilization in HgCl2 (0.1%) for 5 min was used for reducing contamination. MS medium with BAP (5 mg/l) was found to be the best with highest percentage of culture establishment (81.13) and the fastest bud sprout (8.18 days). MS medium supplemented with BAP (4.0 mg/l) and kinetin (0.5 mg/l) resulted in highest shoot proliferation in both the cultivars. The best treatment for micro-shoot elongation was the one where MS medium was supplemented with 0.5 mg/l GA3 giving the highest elongation (3.89 cm). In vitro rooting of micro-shoots was done in half-strength MS medium supplemented with 1.0 mg/l IBA. Hardening was done in glass jar with polypropylene cap.

Rejuvenation influences indirect organogenesis from leaf explants of Pomegranate (Punica granatum L.) ‘Kandhari Kabuli’

ABSTRACTSequential subculturing leads to a gradual physiological change in cells that may be termed ‘rejuvenation’. The effect of repetitive subculturing on callus induction and shoot regeneration from leaf explants of Punica granatum L. ‘Kandhari Kabuli’ were investigated. Surface-sterilised leaves were cultured on 1.0× Murashige and Skoog (MS) medium supplemented with 4.0 mg l–1 α-naphthaleneacetic acid (NAA) and 2.0 mg l–1 6-benzyladenine (BA) for callus induction. Shoots were regenerated from callus on 1.0× MS medium supplemented with 1.5 mg l–1 BA, 0.5 mg l–1 kinetin, and 0.25 mg l–1 NAA. Subculturing of callus onto fresh medium maintained the rate of shoot formation and substantially increased the production of shoot buds up to the second subculture. Following further subculture passages, a lower shoot regeneration potential from callus was observed. A maximum shoot bud induction from callus of 63.9% was observed at the second subculture passage. The rate of multiplication of in vitro shoots increased until the fourth subculture, then became constant. Similarly, in vitro rooting of micro-shoots increased up to the third subculture, followed by a decline during further subculturing.

Micropropagation of <i>Genista aetnensis</i> [(Raf. ex Biv.)DC

Genista aetnensis [(Raf. ex Biv.)DC] is a large deciduous shrub or small tree native to the Italian islands of Sardinia and Sicily. Being winter hardy and characterized by high plasticity in altitude and ecology, the species is grown in gardens and landscaping, both for flower and for its attractive shape. Genista species are generally propagate by seed or semi-hardwood cuttings. In this report an efficient in vitro technique for propagation of G. aetnensis was investigated. Multiple shoots were induced on nodal segments of a mature plant of Genista aetnensis. The Murashige and Skoog medium, augmented with different concentrations of N-6-benzyladenine either singly or in combination with indole-3-acetic acid, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment equal molar concentrations of four cytokinins (2-isopenthenyladenine, kinetin, zeatin and N-6-benzyladenine) were tested for ability to induce axillary shoot development from single node stem segments. The highest rate of axillary shoot proliferation was induced on the medium supplemented with 0.44 µM BA. Growth regulator requirements for shoot proliferation in G. aetnensis were satisfied by BA alone. Explants were divided, subcultured and continued to proliferate shoots. A proliferation rate of 3.5 shoots per single node explants every four weeks occurred. Seven indole-3-acetic acid concentrations (0, 0.23, 0.45, 0.91, 1.82, 3.64 or 7.29 µM) were tested to determine the optimum conditions for in vitro rooting of microshoots. The highest rooting percentage was obtained with indole-3-acetic acid at 3.64 mM (57%). Eighty percent of the in vitro rooted plantlets were successfully established in soil. This micropropagation system of G. aetnensis based on axillary shoot development from nodal segments followed by in vitro rooting should be preferred for rapid and efficient mass propagation of selected clones and could represent an alternative method to sexual and conventional asexual propagation.

Micropropagation of Genista aetnensis [(Raf. ex Biv.)DC

Genista aetnensis [(Raf. ex Biv.)DC] is a large deciduous shrub or small tree native to the Italian islands of Sardinia and Sicily. Being winter hardy and characterized by high plasticity in altitude and ecology, the species is grown in gardens and landscaping, both for flower and for its attractive shape. Genista species are generally propagate by seed or semi-hardwood cuttings. In this report an efficient in vitro technique for propagation of G. aetnensis was investigated. Multiple shoots were induced on nodal segments of a mature plant of Genista aetnensis. The Murashige and Skoog medium, augmented with different concentrations of N-6-benzyladenine either singly or in combination with indole-3-acetic acid, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment equal molar concentrations of four cytokinins (2-isopenthenyladenine, kinetin, zeatin and N-6-benzyladenine) were tested for ability to induce axillary shoot development from single node stem segments. The highest rate of axillary shoot proliferation was induced on the medium supplemented with 0.44 µM BA. Growth regulator requirements for shoot proliferation in G. aetnensis were satisfied by BA alone. Explants were divided, subcultured and continued to proliferate shoots. A proliferation rate of 3.5 shoots per single node explants every four weeks occurred. Seven indole-3-acetic acid concentrations (0, 0.23, 0.45, 0.91, 1.82, 3.64 or 7.29 µM) were tested to determine the optimum conditions for in vitro rooting of microshoots. The highest rooting percentage was obtained with indole-3-acetic acid at 3.64 mM (57%). Eighty percent of the in vitro rooted plantlets were successfully established in soil. This micropropagation system of G. aetnensis based on axillary shoot development from nodal segments followed by in vitro rooting should be preferred for rapid and efficient mass propagation of selected clones and could represent an alternative method to sexual and conventional asexual propagation.

Standardization of protocol for in vitro multiplication of rose (Rosa × hybrida) cv. Happiness

An efficient protocol for in vitro multiplication of Rosa × hybrida L. cv. Happiness was standardized using axillary bud segments. Out of different pre treatments for explants, the highest explant survival (80.25%) was obtained with T1 pre-treatment comprising 0.2% Carbendazim + 0.2% Mancozeb-45 +150 mg/l 8-HQC for 4 hragitation on a horizontal shaker (200 rpm). Sucrose concentration of 30g/l in the medium was found to be optimum for in vitro shoot multiplication. Murashige and Skoog (MS) medium supplemented with 2.5 mg/l BAP + 5.0 mg/l kinetin + 0.1 mg/l NAA + 0.5 mg/l GA3 was found most effective for culture establishment, however, MS medium comprising 2.5 mg/l BAP + 2.5 mg/l kinetin + 0.1mg/l NAA+ 0.5mg/l GA3 along with 40 mg/l adenine sulphate was found to be better for shoot proliferation with highest number of micro shoots (7.10 shoots/explant). Rooting of micro shoots was induced on half strength MS basal medium supplemented with NAA (0.5 mg/l) and IBA (0.5 mg/l) rooting growth regulators. The regenerated plantlets were efficiently hardened in glass jars filled with coco peat + vermiculite + perlite (2:1:1) moistened with half strength MS medium salts and covered with polypropylene lids, thereafter, plants were successfully transferred to the glasshouse with good survival.

To Study the Effect of Activated Charcoal,Ascorbic Acid and Light Duration on InVitroMicropropagation of Aloe vera L

The research was conducted to determine the effect of ascorbic acid (50 mgl-1, 100 mgl-1, 200 mgl-1) and activated charcoal (0.5 gl-1, 1 gl-1, 2 gl-1) independently with different light duration (darkness for 4 weeks, and 16 hours light for 4 weeks followed by 2 weeks in 16 hours light) on shoot regeneration. Explants of Aloe vera were planted on MS basal media supplemented with 1.6 mgl-1 IAA, 4.0 mgl-1 BAP and cultured for 4 weeks. After 4 weeks, degree of explant browning was evaluated. Explants were then cut vertically into two pieces and planted on shoot regeneration media. After 4 weeks more number of shoot developed on shoot regeneration medium, i.e. MS medium supplemented with 1.6 mg/l IAA + 4.0 mg/l BAP + 100 mg/l ascorbic acid + 0.5g/l activated charcoal. Browning of ex- plants was minimized in this medium and elongation of micro-shoots and the growth of the plantlets were also better. Further elongation and rooting of micro-shoots were obtained when sub-cultured on to MS basal medium containing 0.5g/l activated charcoal and 100% of the survival of rooted plantlets was observed after acclimatization. Therefore, the above protocol could be effectively used in rapid mi- cropropagation of elite plants of Aloe vera.

Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania somnifera L.

An improved and methodical in vitro shoot morphogenic approach through axillary bud multiplication was established in a drug yielding plant, Withania somnifera L. Effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyladenine (2iP), and thidiazuron (TDZ)] either singly or in combination with α-napthalene acetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium were tested. The highest regeneration frequency (90 %) with optimum number of shoots (32 ± 0.00)/explant were obtained on MS medium fortified with 2.5 μM 6-benzyladenine (BA) and 0.5 μM NAA and 30 g/l sucrose at pH 5.8. Among the tried TDZ concentrations, 0.5 μM resulted in maximum number of shoots (20.4 ± 0.40)/explant after 4 weeks of exposure. The proliferating shoot cultures established by repeated subculturing of the mother explants on the hormone-free medium produced the highest shoot number (29.4 ± 0.40) with shoot length (6.80 ± 0.12 cm)/explant at fourth subculture passage, which a decline in shoot proliferation was recorded. Different concentrations of NAA were tested for ex vitro rooting of microshoots. The maximum percentage of rooting 100 % with maximum roots (18.3 ± 0.1) was achieved in soilrite when basal portion of the microshoots were treated with 200 μM (NAA) for 15 min per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. The PCR-based single primer amplification reaction (SPAR) methods which include random amplified polymorphic DNA (RAPD) and direct amplification of minisatellite DNA (DAMD) markers has been used for assessment of genetic stability of micropropagated plantlets. No variation was observed in DNA fingerprinting patterns among the micropropagated and the donor plants illustrating their genetic uniformity.

Direct adventitious shoot bud formation on hypocotyls explants in Millettia pinnata (L.) Panigrahi- a biodiesel producing medicinal tree species.

A reproducible protocol developed for in vitro regeneration of Milletia pinnata using hypocotyl segments. Multiple shoots were induced from hypocotyl explants through direct adventitious shoot bud regeneration. The proximal end of hypocotyls was responsive for shoot bud induction. Silver nitrate and adenine sulphate had a positive effect on shoot bud induction and elongation. The maximum response and number of shoot bud produced in media supplemented with 8.88μM BAP with 108.6μM adenine sulphate and 11.84μM silver nitrate. Elongated shoots were harvested and successful rooting of microshoots achieved on MS media supplemented with 9.84μM IBA, with 81.1% rooting. Remaining shoot buds sub-cultured for further multiplication and elongation. Each subculture produced eight to nine elongated microshoots up to four subcultures. The rooted microshoots were successfully hardened and transferred to field.

English

Chlorophytum borivilianum Sant. et Fernand. is an endangered herb, the tuberous roots of which are source of medicinally important steroidal saponins. In the present study, propagation of C. borivilianum using a bench top stirred bioreactor with liquid medium via multiple shoot culture has been reported. One week old shoots along with shoot base part (1.5 cm) obtained from shoots regenerated in vitro in liquid medium shake flasks containing 22.2 µM 6-benzylaminopurine, were used as explants. An inoculum density of 120 explants/2.5 L liquid Murashige and Skoog medium supplemented with 22.2 µM 6-benzylaminopurine was found optimal for shoot growth. After three weeks of culture, 4.4-fold increase in biomass (fresh weight) was obtained. Shoots regenerated in bioreactor were rooted ex vitro on three-fourth strength liquid MS medium supplemented with 9.8 µM indole-3-butyric acid. Plantlets with 100% rooting of microshoots were hardened and established in the glasshouse with 85% survival rate. Due to rapid and efficient propagation in bioreactor with high survival rate, this protocol may be employed for conservation and large-scale multiplication of C. borivilianum . Keywords: Bioreactor, Chlorophytum borivilianum , hyperhydricity, saponins, shoot culture African Journal of Biotecnology , Vol 13(17), 1772-1778

Diversity among wild accessions of Bacopa monnieri (L.) Wettst. and their morphogenetic potential

Biochemical and molecular diversity among 14 accessions of Bacopa monnieri (L.) Wettst. collected from various locations of India was investigated. A significant variation was recorded in bacoside A contents of these accessions. A scatter plot of principle component analysis based on bacoside A contents clubbed these populations into two major groups and accession BM14 was placed separately. Similarly, about 35 % variations were detected in these populations based on combined data of random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR). Individually, ISSR markers detected higher variation (44.9 %) as compared to RAPD markers (23 %). Clustering based on molecular marker data grouped these accessions into two major groups and also placed accession BM14 as an out group. The shoot organogenic potential of leaf explants taken from microshoots and rooting of microshoots also varied among accessions. Maximum shoot organogenic potential was observed in accession BM5 and maximum rooting potential was observed in accessions BM1, BM2, BM7, BM10 and BM14. Present study is an important step for developing long-term strategy for conservation of this important medicinal herb.