Half-strength Medium Research Articles

Indirect organogenesis in Ephedra foliata

Ephedra foliata Boiss. ex C.A. Mey (Ephedraceae) is a well-known source of the alkaloid ephedrine used for the treatment of chronic asthma and associated respiratory ailments. This investigation was conducted to standardize an efficient regeneration protocol for E. foliata via indirect organogenesis through intermodal explants. Maximum callus induction (80.3%) was achieved on Murashige and Skoog (MS) medium supplemented with 0.5 mg L‑1 2,4‑dichlorophenoxyacetic acid + 0.5 mg L‑1 kinetin and additives (50 mg L‑1 ascorbic acid and 25 mg L‑1 citric acid). The maximum shoot regeneration (n = 5.27) was achieved on MS medium containing 6.0 mg L‑1 kinetin, followed by 5.0 mg L‑1 6‑benzylamino purine (n = 4.27). MS half strength medium with 3.0 mg L‑1 α‑naphthalene acetic acid resulted in the highest rooting percentage (32%). Sixty percent of the plantlets survived during acclimatization and were successfully transferred under field conditions. These plants and callus will be a suitable source of plant material for mass multiplication, genetic modification for enhanced bioactive constituents, and germplasm conservation.

Chitosan as a Growth Promoter and Enhance Survival Rate in an in vitro Culture of Banana (Musa spp.) Cultivar ‘Bantala’

Background: Banana (Musa spp.) is one of the most consumable fruits and cultivated around the globe. It contains high nutritional value as well as the high demand of the market. The microbes are the main problem for the propagation of banana plants in tissue culture. Chitosan is one of the best substances for the eradication of contamination and also growth stimulators of banana plants. This study is based on the micro-propagation of the bantala variety of Musa species and free from microbe infection. Methods: The rhizome and sucker as explants of Musa cv. Bantala. The different combination concentrations of 6-Benzylaminopurine (BAP), indole-3-acetic acid (IAA) and chitosan (CS) were tried in Murashige and Skoog medium for in vitro response of plants, shoot initiation and shoot proliferation. The formation of rooting was used as the half-strength Murashige and Skoog (MS) medium with indole-3-butyric acid (IBA) and chitosan (CS). Result: The best response, shoot initiation and shoot proliferation were observed at 6-Benzylaminopurine (5.0 mg/L)+indole-3-acetic acid (0.5 mg/L)+chitosan (25 mg/L) and 6-Benzylaminopurine (4.0 mg/L)+indole-3-acetic acid (0.5 mg/L)+chatoyant (25 mg/L) in both of rhizome and sucker respectively. The maximum root formations were observed in the medium containing half-strength Murashige and Skoog medium+1.0 mg/L indole-3-butyric acid+25 mg/L chitosan in the rhizome and 0.8 mg/L indole-3-butyric acid+25 mg/L chitosan in the sucker. The successful survival rate of sucker and rhizome under the acclimatization condition was recorded as 90% and 88% compared with control as 66% and 63% respectively. This standardized protocol might be useful for the mass production of bantala variety as well as other cultivars of banana plants.

Особенности индуцированного морфогенеза и регенерации растений из соматических тканей люцерны сортов отечественной селекции в культуре in vitro

The authors have studied Morphogenesis and regeneration features of domestic breeding cultivars of alfalfa and conditions for obtaining regenerant plants from the morphogenic callus tissues. As a result of the study, the efficient sterilization protocol of alfalfa seeds was established with the use of sterilizing agents as 70% ethanol and 10% sodium hypochlorite which resulted in the production of 100% aseptically viable seedlings for the further in vitro studies. Callus initiation from cotyledons and hypocotyls was implemented on a Gamborg B5 nutrient medium containing 2,4-dichlorophenoacetic acid (2,4-D), kinetin in a concentration of 5,0 mg/l, and naphthaleneacetic acid (NAA) of 0,1 mg/l. Induction of morphogenic callus was achieved on a Gamborg B5 medium with 0,2 mg/l 6-benzylaminopurine (BAP). Further shoot initiation occurred on a hormone-free Gamborg B5 medium with the addition of Gamborg B5 vitamins, and on a half-strength medium with the addition of 0,2mg/l kinetin. It was found that established method of obtaining regenerated plants from the callus cultures of Medicago Varia proved itself effective.

Establishment of Codonopsis pilosula (Franch.) Nannf. transformed roots, influence of the culture conditions on root growth and production of essential oil

Codonopsis pilosula (Franch.) Nannf. is a species whose radix has long been used in traditional folk medicine in China, Japan and Korea. Codonopsis Radix is obtained from cultivations mainly in Gansu, Shanxi, Sichuan, and Guizhou Provinces of China. This study describes an efficient protocol for genetic transformation of C. pilosula by Rhizobium rhizogenes and the obtainment of transformed roots of this species with valuable medicinal properties. It was found that both the bacterial strain (A4 and ATCC 15834) and acetosyringone (AcS) addition influenced the efficiency of transformation. The highest frequency of root induction (69 %) was observed on the Murashige and Skoog (MS) agar medium supplemented with AcS after transformation with A4 strain. The transformation was confirmed by PCR analysis. Of the tested media (MS, Schenk and Hildebrandt - SH, Gamborg - B5, and Woody Plant - WPM) those with full macro- and microsalt concentrations elicited significantly greater C. pilosula transformed root (CP2) biomass accumulation than the half-strength media, apart from MS medium. The greatest CP2 root biomass was observed in SH medium (1.65 g flask−1 DW). Among the tested sucrose concentrations (10−70 g L−1), the best result was obtained by using 30 g L−1 sucrose. Essential oils isolated by hydrodistillation from CP2 transformed roots and commercially available raw material, C. pilosula roots, had very similar qualitative composition. The most important group of common constituents of both oils were sesquiterpene hydrocarbons. The main among twenty two compounds in this group possessed skeletons of barbatane, thujopsane and bisabolane; the predominant ones being α- and β-barbatene, followed by β-chamigrene and thujopsene. This study presented that C. pilosula transformed roots can be a potential source for the industrial production of the raw material from species of the genus Codonopsis used in traditional Chinese medicine.

English

Developing a protocol for somatic embryogenesis (SE) for adult mother plants of Jatropha curcas, aids to overcome problems such as harvesting time and an uneven yield, providing the opportunity to propagate proven elite genotypes. Until now, several authors have achieved SE in J. curcas using different explants, however, in none of these protocols adult plants have been used as mother plant material. Furthermore, the challenge of overcoming the morphogenesis limitations successfully and further regeneration of adult plants of J. curcas has been great. The main objective of this study was to develop a somatic embryogenesis protocol for adult plants of the oil producing J. curcas. transverse thin cell layers (tTCL) of young petioles were used as explants. Half strength Y3 and MS media were used containing three different concentrations of 2,4-dichloropherioxyacetic acid and 6-benzil-aminopurine in a factorial trial. Nine accessions of J. curcas of various ages (3-12 years.) were tested using this protocol. All accessions responded positively to callus induction and to the induction of somatic embryos, demonstrating the protocol to be genotype-independent. This study enables micropropagation of adult of proven elite plants of J. curcas via somatic embryogenesis.    Key words: Jatropha curcas, transverse thin cell layers (tTCL), somatic embryos, half strength media, regeneration.

In vitro propagation and analysis of mixotrophic potential to improve survival rate of Dolichandra unguis-cati under ex vitro conditions

An efficient and reproducible protocol for in vitro propagation of Dolichandraunguis-cati has been established for the first time from nodal segments. In order to enhance survival rate under ex vitro conditions, photosynthetic potential of in vitro grown plantlets was also studied through JIP test based analysis of polyphasic OJIP chlorophyll a fluorescence OJIP transients, density of active reaction centers, light harvesting efficiency, electron transfer rate, dissipation energy, maximum quantum yield of primary PSII photochemistry and photosynthetic performance index. The best morphogenetic in term of explants response (92.2 %), shoot number (3.43 ± 0.07) and shoot length (4.7 ± 0.31 cm) was obtained on Murashige and Skoog medium supplemented with 0.5 mg l−1 BAP and 1.0 mg l−1 TDZ. The shoots exhibited high frequency rhizogenesis on half strength medium augmented with 2.0 mg l−1 IAA. In vitro plantlets developed highest rate of photosynthesis on day 18 after the initiation of rhizogenesis. High survival rate (96.16%) under ex vitro conditions was observed when in vitro plantlets having high photosynthetic efficiency (Fv/Fm > 0.75) were subjected to hardening and acclimatization process. Plantlets with reduced photosynthetic performance exhibited low survival rate under natural conditions. The developed in vitro protocol will be useful for genetic improvement and multiplication of D. unguis-cati. The results of this study also show that photosynthetic screening of in vitro developed plantlets is highly essential after the rhizogenesis process to achieve higher survival rate under field conditions.

Effect of different growth regulators on in vitro regeneration of cotyledon explants of rough lemon (Citrus jambhiri Lush.) via callus induction

In order to establish in vitro protocols for raising aseptic cultures of cotyledon segments excised from five-week-old in vitro raised seedlings were used as explant material. Among surface sterilants, mercuric chloride 0.1% for three minutes provided least rate of contamination (24%) and the maximum explant survival (76%). The highest per cent culture establishment of 97.79% was obtained in cotyledon explants on Murashige and Skoog (MS) containing 2,4-D (3.0 mg/l) and maximum callus induction (95.41%) was observed on MS medium supplemented with 2,4-D (2.0 mg/l). Among different plant growth regulator treatments, maximum callus regeneration of 82.19% was observed on MS medium supplemented with BAP (3.0 mg/l) whereas, minimum callus regeneration (4.33%) was found in MS medium supplemented with BAP (3.0 mg/l) in combination with IAA (0.75 mg/l) and malt extract (500 mg/l). Maximum rooting response (96.00%) was recorded on MS (half strength) medium with NAA (2.0 mg/l) and was at par with rooting percentage of 92.00% on MS (half strength) medium supplemented with NAA (1.5 mg/l). The plantlets were successfully acclimatized in different potting mixtures and highest survival rate (91.35%) was achieved in potting mixture containing garden soil with sand and farmyard manure (1:1:1).

Trials on In vitro Propagation and Using Natural Additives for Myrtus communis L. Plant

Explant types, anti-oxidant pre-treatments, organic additives, natural additives, vitamins mix strengths, cytokinin types and concentrations, different medium strengths, GA3 concentrations, auxin types and concentrations were studied during the period from 2017 to 2018 to establish a protocol for in vitro propagation of Myrtle. It was found that culturing of pre-treated shoot tips with anti-oxidant solution (A.O.S) on modified Murashige and Skoog (MS), or Murashige and Skoog medium supplemented with PVP as anti-oxidants induced the best results in reducing free phenolic compounds and enhancing explant development parameters. Also, adding combination of tryptophan, adenine sulphate and coconut water as organic additives maximized survival percentage and improved explant development. In the same time, adding combination of coconut water at 5% plus Banana pulp plus Papaya extract at 50 g/L of were helpful in maximizing number of shoots/plant, shoot length and greening parameters. Also duplicating the dose of vitamin mix of Gamborg medium improved explants development and survival (%) of explant. Meanwhile, using of 1.0 mg/ L BAP increased proliferation. Meanwhile, addition of 2.0 mg/ L GA3 to half strength medium maximized shoot length. Moreover, the addition of 2.0 mg/L IBA to the culture medium induced the highest number of roots/plant.

Development of an Embryo Rescue Protocol for Butterfly Weed

We documented a successful embryo rescue (ER) protocol for butterfly weed (Asclepias tuberosa), a member of the milkweed family (Asclepiadaceae). Milkweed (Asclepias sp.) includes more than 100 species native to the United States, is an important pollinator plant, and has many commercially desirable traits. However, there is little commercial production outside of native plant nurseries because milkweed species are typically seed-grown and suffer from low seed set during pollination, late-term abortion of seed pods, and nonuniform germination. This project determined the optimal growing media (study one) and embryo maturity (study two) to recover mature seedlings from excised embryos and compared the results to those of traditional methods of seed germination (in soilless substrate). Study one investigated three different media: Murashige and Skoog (MS) medium at full strength and half strength and woody plant medium. MS medium at half strength was optimal for butterfly weed germination and maturation, with greater root and shoot lengths at the time of harvest. In study two, the effects of MS medium at half strength on embryo maturation 90, 60, and 30 days after pollination (DAP) were investigated. The optimal time to harvest embryos was 60 DAP; embryos at 30 DAP were capable of germination but not maturation. A mean germination rate of 97.4% was observed when using embryo rescue, but it was 72.3% with mature seed germinated in soilless substrate typical of commercial production. A similar increase in germination rates was observed for all embryo maturities when compared with seed germinated using soilless substrate. The protocol developed for this study should help to standardize production, reduce propagation time, and improve the commercial acceptance and profitability of milkweed.

Effect of different PGRs on in vitro organogenesis in Viola canescens Wall. ex. Roxb. from petiole callus culture

An efficient protocol for in-direct in vitro regeneration of Viola canescens is described using petiole derived callus. Proliferation of callus was achieved using 2,4-D, 2,4-D: Kn, 2-4D: BAP; IBA alone or in combination with Kn on Murashige and Skoog medium. The callus differentiated into multiple shooting buds on MS medium fortified with different concentrations of various cytokinins (Kn, BAP, Zeatine and Gibberellin). Maximum average number of shoots (19.93 ± 1.79) and maximum average length of petiole (5.41 ± 0.47) was achieved when medium was fortified with 22.20 µM BAP and 18.60 + 4.0 µM Kn + Gibberellin, respectively. The microshoots were separated and rooted on full and half strength MS medium fortified with different concentrations of various auxins (IBA and NAA). Maximum average number (14.30 ± 2.24) and length (3.98 ± 0.73) of root was recorded on half strength medium fortified with 4.92 µM IBA. The regenerated plantlets were phenotypically normal and successfully transplanted to soil conditions with 90% survival percentage.

In vitro mass propagation and conservation of a rare medicinal plant, Zhumeria Majdae Rech.f & Wendelbo (Lamiaceae)

Abstract The monotypic Zhumeria majdae (Lamiaceae), an endangered medicinal and aromatic plant in the south part of Iran, has a low propagation rate in natural condition and therefore an efficient method for the in vitro-propagation is required. In the present study, the effects of source and position of the explants as well as various concentrations of 6-benzylaminopurine and kinetin on inhibition of hyperhydricity and in vitro mass propagation of the plant were investigated. The best shoot formation (1.9 ± 0.07) was obtained with a Murashige and Skoog medium fortified with 17.7 µM 6-benzylaminopurine. Regenerated shoots, elongated on the MS medium containing 2.2 μM 6-benzylaminopurine, were rooted on the different tested media, with the most abundant (68.6 ± 4.1%) and strongest roots obtained on half-strength medium without plant growth regulators. Hydro-distilled essential oils of in vitro regenerated plant were analyzed by gas chromatography–mass spectrometry and compared with the essential oil of wild plant. Thirty and twenty compounds representing 98.2% and 98.7% of the total oils were identified in the oils of wild plant and in vitro regenerated plant, respectively. The major essential oil components were linalool (31.7% and 41.7%), camphor (28.0% and 32.4%), limonene (4.6% and 8.3%), camphene (4.1% and 3.5%) and E-caryophyllene (1.0% and 2.7%) in the studied (wild plant and in vitro regenerated plant) essential oils. The in vitro regeneration system could be utilized for both conservation, large-scale multiplication and production of rich linalool essential oils of Z. majdae.

EFFECT OF SUCROSE AND MEDIA STRENGTH ON IN VITRO MULTIPLICATION IN SWERTIA CHIRATA BUCH.-HAM EX WALL: AN ENDANGERED MEDICINAL HERB

Objective: The present study was performed to investigate the role of varying concentrations of carbohydrate source and strengths of nutrient medium in growth and development of in vitro shoots of Swertia chirata-an endangered medicinal plant.Methods: MS medium supplemented with 6-Benzylaminopurine (4.44 µM), Indole-3 acetic acid (2.85 µM) and Adenine sulphate (271.45 µM) was used to test the efficiency of of sucrose at concentrations of 1-5% and of media strength varying from full to one-fourth. The data were analysed using analysis of variance (ANOVA) of Completely Randomized Design (CRD) in GenStat 5 Edition 3.2 for PC/Windows NT (Copyright 1995, Lawes Agricultural Trust (Rothamsted Experimental Station))Results: Observations on axillary shoot multiplication indicated that sucrose at a concentration of 3% and MS medium in its full strength proved to be most optimal for in vitro culture multiplication. On this medium combination mean number of 11.80 shoots (after 4 w) and 18.50 shoots (after 8 w) could be obtained On sucrose free medium the shoots exhibited necrosis while at lower concentrations of 1-2% sucrose, the shoots developed were thin and unsuitable for further growth in vitro. At higher levels of sucrose in the medium, the shoots became thick and stunted. Similarly, reduction in medium strength resulted in a decline in shoot number and shoot length to an average of 6.50 shoots (1.33 cm mean length) on half strength medium and 5.60 shoots (0.88 cm mean length) on one-fourth strength; as observed after 4 w.Conclusion: The experimental findings suggest that any decline from the standard had a significant effect on the number, size and overall health of shoots developed in vitro. The conditions so standardized augment the production of healthy shoots that shall aid in subsequent rooting and survival after transplantation of tissue-culture raised plantlets.

IN VITRO REGENERATION AND MASS PROPAGATION OF AZIMA TETRACANTHA [LAM.] FROM THE LEAF EXPLANTS THROUGH CALLUS CULTURE

In the present study the protocol for callus induction and regeneration in Azima tetracantha has been developed in culture medium. The young apical leaf explants were used for callus induction on MS medium containing BAP and NAA at 1.0 and 0.4mgl-1 respectively showed maximum callus induction (73%). The amount of callus responded for shoot formation (74%) was obtained in the MS medium containing BAP (1.5 mgl-1) and NAA (0.3mgl-1).The elongated shoots were rooted on half strength medium supplemented with IBA (1.5 mgl-1) and Kn (0.4 mgl-1) for shoots rooted. Regenerated plantlets were successfully acclimatized and hardened off inside the culture and then transferred to green house with better survival rate.

In vitro propagation and conservation of an endangered high value medicinal herb Swertia chirayita of temperate Himalayas

The current study reports on in vitro propagation and conservation of critically endangered medicinal plant Swertia chirayita. The sterilized explants (leaves) were cultured on Murashige and Skoog’s (MS) medium supplemented with 0.1 mg/L naphthalene acetic acid (NAA) and 3.0 mg/L benzyl adenine (BA) for callus induction, the same medium was used for shoot regeneration and for direct shoot regeneration from in vitro leaves. For in vitro multiplication shoots were cultured on MS medium supplemented with 2.5 mg/L benzyl adenine and 0.1 mg/L Kinetin (Kn). MS half strength medium supplemented with 400 mg/L activated charcoal and 0.1 mg/L naphthalene acetic acid showed 80.30% root induction from in vitro grown shoots. The in vitro raised plantlets were successfully acclimatized. All the regenerated plantlets appeared normal with 70–80% survival rate, while In vitro conservation was carried out by using two different approaches namely slow growth by changing media composition (sucrose and abscisic acid) at low temperature and cryopreservation following vitrification. With increase in concentration of sucrose and ABA decrease in growth of in vitro shoots was observed. At low temperature the in vitro shoots incubated at 4 and 10 °C both showed 100% retrieval. During studies the vitrified shoot gave retrieval of 42.33% when pre-cooled at 4 °C while only 22.77% vitrified shoots were retrieved from those pre-cooled at 10 °C.

Optimizing culture conditions for establishment of hairy root culture of Semecarpus anacardium L.

Semecarpus anacardium L. is a tree species which produces secondary metabolites of medicinal importance. Roots of the plant have been traditionally used in folk medicines. Different strains of Agrobacterium rhizogenes (A4, ATCC15834 and LBA 9402) were used for induction of hairy roots in in vitro grown tissues of the plant. Hairy root initiation was observed after 25–30 days of infection. Optimum transformation frequency of 61% was achieved on leaf explants with ATCC15834 strain. Infection time of 30 min resulted in greater transformation frequency compared to 10 and 20 min, respectively. The hairy roots cultured in growth regulator-free semi-solid woody plant medium differentiated into callus. Whole shoots infected with ATCC 15834 were found to produce more transformants upon co-cultivation for 4 (65%) and 5 (67%) days. Induction of hairy roots in stem explants infected with ATCC 15834 was lower (52%) compared to leaves (62%) after 4 days of co-cultivation. In A4 and LBA9402 strains transformation efficiency was 49 ± 2.8% and 36 ± 5.7% in shoots after 4 days of co-cultivation. Transformation frequency was higher in ATCC15834 strain, irrespective of explants. The hairy roots of S. anacardium elongated slowly upon transfer to half-strength liquid medium. After 3–4 passages in liquid medium slender hairy roots started differentiating which were separated from the original explants. Visible growth of the roots was observed in hormone-free liquid medium after 2–3 months of culturing. Polymerase chain reaction with gene-specific primers from rol A, B and C genes confirms the positive transformation events.

Embryogenic Callus Induction from Leaf Tip Explants and Protocorm-Like Body Formation and Shoot Proliferation of Dimorphorchis lowii: Borneon Endemic Orchid

Dimorphorchis lowii is a rare epiphytic orchid endemic to Borneo and very popular due to its ornamental value. For the purpose of mass propagation, procedures for in vitro propagation through callus were developed. The objectives of this study were to determine the effects of plant growth regulators (PGRs) on callus induction from leaf tip explant and the effects of complex additives on PLBs formation and shoot development . The best combination of PGR was 3.0 mg L -1 Thidiazuron (TDZ) with 0.046 mg L -1 NAA supplemented in half-strength medium (½ MS) (Murashige & Skoog, 1962). The percentage of survival and callus formation obtained were 96.0 % ± 19.8 and 52.0 % ± 16.5 respectively. Maximum shoot proliferation from PLBs was observed in Knudson C (KC) medium enriched with 15 % (v/v) coconut water. In this treatment, 10.2 ± 6.2 shoots were produced from one callus explant. Shoots were rooted on KC medium containing 15 % (v/v) coconut water and transferred in a medium mixture containing sphagnum moss, charcoal, brick and coco peat with the ratio of 1:1:1:1. After 2 months being acclimatized in the glasshouse, 78 % of plantlets survived. Histology observations showed that embryogenic callus derived from leaf tip explants might originate from epidermis and mesophyll cells and was capable of developing into complete plantlets.

Tissue Culture Studies on Propagation of Some Ornamental Plants 1. Starting and Shooting Stages of Phytolacca dioica L.

This work was carried out in the Tissue Culture Laboratory, Horticulture Research Institute, Agricultural Research Center, Giza, Egypt from the period elapsed from 2014 to 2016 to study the effect of sterilization on propagation of Phytolacca dioica, L.. Results of this study could be briefed in the following outlines: Chlorox concentrations at 30 or 35 % gave the highest survival percentage of Phytolacca dioica, L.. In addition, soaking explants in the sterilant solution for 20 or 25 minutes resulted in the highest survival percentage. Similarly, the interaction treatment demonstrated that using chlorox at 35% for 25 minutes resulted in the highest survival%. In the multiplication stage it was found that using BAP at 10 ppm resulted in the highest number of shoots and leaves. BAP at 6 or 8 ppm induced the highest content of total chlorophyll. The longest shoots were a result of applying BAP at 3 ppm. Inoculating explants on half strength medium resulted in the highest values of number of shoots and leaves, as well as highest contents of total chlorophyll and carotenoids. MS medium at 3 quarters strength shared in the first position of number of leaves. The longest shoots were a result of using MS medium at full strength. The interaction between BAP levels and MS medium strength, indicated that using MS medium at half strength supplemented with BAP at 10 ppm produced highest number of shoots. The same medium strength with BAP at 8 or 6 ppm gave the highest contents of total chlorophyll and carotenoids. MS medium at 3 quarters strength combined with PAP at 10 ppm gave highest number of shoots and leaves. MS medium at full strength plus BAP at either 3 or 10 ppm resulted in the highest shoot length and number of leaves.

Efficient in vitro plant regeneration from seedling-derived explants and genetic stability analysis of regenerated plants of Simarouba glauca DC. by RAPD and ISSR markers

Simarouba glauca DC. is a multipurpose tree species known for oil, timber, and medicinal properties. The application of biotechnological methods for genetic improvement of this species depends on the availability of an efficient plant regeneration system. In this study, the shoot regeneration potential of various seedling-derived explants was assessed after culturing on Murashige and Skoog (MS) and woody plant (WP) medium containing different growth regulators. The explants differed in their capacity for shoot bud formation and subsequent shoot elongation on the media tested. Shoot bud induction was achieved at a high frequency (44.8–76.2%) from different explants on MS medium with 2 mg L−1 6-benzylaminopurine (BAP) as compared to other media tested. Cotyledons exhibited the highest capacity for shoot bud induction (76.2%) and shoot elongation (9.1 elongated shoots per explant). The in vitro-regenerated shoots rooted at a frequency of 66.7% after pulse treatment in 10 mg mL−1 indole-3-butyric acid (IBA) solution for 5 min followed by culture on half-strength WP medium with 0.2 mg L−1 IBA. The regenerated plants were acclimatized and established in the glasshouse with a survival rate of 80%. Molecular characterization of regenerated plants using 14 random amplified polymorphic DNA (RAPD) and 15 intersimple sequence repeat (ISSR) primers revealed a high number of monomorphic bands, with only 1.6–2.6% of the bands being polymorphic. The regeneration system established in the study has the potential to be used for rapid multiplication, conservation, and genetic transformation of this species.

In vitro Culture and Biochemical Attributes of Bergenia ciliata (Haw.) Sternb.

Bergenia ciliata is one of the medicinally important and vulnerable plants of Kashmir Himalaya. A highly reproducible tissue culture protocol of B. ciliata vis-a-vis evaluating its biochemical attributes was studied. The best response for callus formation was obtained on Gamborg’s half strength medium with thidiazuron (2 mg/l) and 1-napthlene acetic acid (2 mg/l) after 6 weeks. Maximum number of 54 shoots were obtained on B5 ½ strength medium + 6-benzylaminopurine 2 mg/l + indole-3-acetic acid 1 mg/l. The complete plants were acclimatised in lab and then transferred to green house conditions with survival rate of 65 %. The in vitro raised, wild plant leaves and callus of B. ciliata were evaluated for biochemical attributes (chlorophyll, carotenoids, total carbohydrates and total soluble protein). Total carbohydrate content was higher in wild plant leaves (15 mg/g) as compared to in vitro plant leaves (11.7 mg/g). Chlorophyll a, b, total chlorophyll and carotenoid content was found to be 0.15, 0.05, 0.24, 0.10 mg/g respectively in wild grown leaves and 0.11, 0.05, 0.15 and 0.06 mg/g respectively in leaves of in vitro raised plants. Carotenoid content, however, was observed significantly (P < 0.005) higher (0.22 mg/g) in callus. Soluble protein content was found to be 142.5 mg/g fresh weight in wild plant leaves but it was 157.5 mg/g fresh weight in in vitro plant leaves. In current study, reproducible in vitro regeneration protocol was developed. It was also observed that in vitro culture had significant influence on expression of primary metabolites vis a vis plant growth.

Employment of Tissue Culture Techniques in Improvement Propagation of Paulownia Tomentosa Plant

Different media and explant types, anti-oxidant pre-treatments, additives, cytokinin types &concentrations as well as medium strength, GA3 concentrations, auxin types &concentrations were studied to find out the best protocol of in vitro propagation of Paulownia tomentosa. The results indicated that culturing of pre-treated shoot tips with anti-oxidant solution on modified Murashige&Skoog medium supplemented with PVP and activated charcoal as anti-oxidants, as well as adenine sulphate and coconut milk as additives maximized survival percentage and improved explant development. Also, using of 2mg/ L BAP increased proliferation. Addition of 1.0 mg/ L GA3 to half strength medium maximized shoot length. Moreover, addition of 1.0 IBA to the culture medium encouraged the highest shoot length and number of roots.