Agar-solidified MS Medium Research Articles

An Efficient Micropropagation Protocol For Mucuna Pruriens (L.) Dc.

An efficient protocol was developed for rapid micropropagation of Mucuna pruriens (L.) DC., an important medicinal plant species of Bangladesh. For the purpose, leaf segments, nodal segments and shoot apices of in vitro grown seedlings and cotyledon segments of immature seeds of field grown plants were cultured on agar solidified MS medium supplemented with different combinations of PGRs (Plant Growth Regulators). Only leaf and cotyledon segments produced light green to green friable/ compact callus in some of the media combinations. Combination and concentration of PGRs played important role in induction of such tissue. The induced tissues failed to differentiate on further culture in a wider spectrum of PGR supplemented media. On the other hand nodal segments and shoot apices produced multiple shoot buds when grown on a wide range of PGRs (BAP, NAA, IAA, Kn) supplemented media. Maximum number of multiple shoot buds were produced in medium containing 1.5 mg/l BAP + 1.0 mg/l IAA. The elongated shoot buds on further culture in rooting media produced strong and stout root systems. Half strength MS media fortified with either 1.0 mg/l IAA or 2.0 mg/l IAA was most effective for induction and proliferation of roots. These complete plantlets were successfully acclimatized and established in outside pots through a successive phase of acclimatization. The Chittagong Univ. J. B. Sci.,Vol. 7(1 &2):79-89, 2012.

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

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

Ex-situ Conservation of Bulbophyllum leopardinum, A Threatened Medicinal Orchid of Nepal

A successful micropropagation method was developed via the in-vitro seed germination and seedling growth of the epiphytic and/or lithophytic orchid Bulbophyllum leopardinum, a species having horticultural and therapeutic significance. To enhance seed germination, several quantities and combinations of naphthalene acetic acid (NAA), 6-benzyl amino purine (BAP), indole acetic acid (IAA), gibberellic acid (GA3), and coconut water (CW) were added to 0.8% (w/v) agar-solidified MS medium. Half-strength MS medium has been experimented with alone and in combination with BAP, Kinetin (Kn), and GA3 to promote shoot development. In-vitro-developed healthy shoots were chosen to establish roots in a half-strength MS (HMS) medium supplemented with various auxins. The best and earliest seed germination with the greenest protocorms (96.3±0.5% in 7 weeks) was achieved on HMS medium fortified with 15% CW (H15C). Further tests for the shoot as well as root development were continued with an H15C medium. H15C with 1 mg/l BAP and 1.5 mg/l Kinetin was most effective for early in vitro development and differentiation into seedlings with the many long shoots (9.3±0.1 shoots and 2.4±0.1 cm per culture) within 12 weeks of sub-culture. The most suitable rooting hormone was 1 mg/l NAA (4.2±0.26 roots per culture). This medium also produced the longest roots (1.9±0.09 cm per culture). By successfully developing a protocol for the mass propagation of B. leopardinum, this research has enhanced both the cultivation and the commercialization potential of this species.

PROPAGATION PROTOCOL OF THE MEDICINAL PLANT – ALOE VERA USING TISSUE CULTURE

Aloe vera is one of the most popular cactus-type plants in the global market due to its widespread uses in pharmaceutical, cosmetic, food, and decorative purposes. The present study derived callus cultures from the Aloe vera plant leaves, then reproduced on agar-solidified MS medium from June to December 2021 at the University of Mosul, Iraq. Results revealed that the MS medium + 3.0 mg L-1 benzyl adenine (BA) proved suitable for induction of leaf callus up to 85%, while the MS medium supplement with 1.0 and 2.0 mg L-1 BA reached 70%. The MS medium with 1.0 mg L-1 BA showed the best results for growing apical shoots of A. vera plants and producing vegetative branches. The formation of roots emerged within two weeks after placing them on the rooting medium. The shoots regenerated from the growing apices and were rooted easily in agar-solidified MS medium. The obtained plants attained successful acclimatization in terms of their growth and length, afterward, transferred to the peat-moss mixture.

Application of multiple strategies to efficiently break seed dormancy of permanently odd pentaploid rose hip (Rosa canina l.) under in vitro conditions

Rosehip (Rosa canina L.) is an important medicinal, and ornamental plant species with high commercial value. Its sugars, phenolics, organic acids, water-soluble vitamins, and mineral contents composition varies depending on environmental conditions and genetics. The plant is also used as a perfect rootstock for many rose cultivars. Seed proliferation is extremely difficult because of multiple endogenous and exogenous dormancy factors. There is a need to breed standard rosehip cultivars rootstock developments with desired characteristics and outperforming yields in fields. The study aimed to break the seed dormancy of rosehip under in vitro conditions by application of multiple strategies in an efficient manner. The seeds were treated with different doses of GA₃, scarified mechanically, stratified on agar solidified MS medium containing GA₃ singly or in combinations of the two each or three treatments, and subjecting them to the regulated physiological treatment of alternating warm/chilling and cold/dark treatments in parallel for 21 d ensued by 18 d warm/light treatments. It was noted that the rosehip seeds could be germinated variably if the scarified seeds were stratified on agar solidified MS medium with or without GA3. Optimum seed germination (80.00- 85.00%) was noted when the three treatments were combined and the seeds were subjected to regulated and alternating warm and chilling treatments for 21 d leaving them for 18 d in warm/light. These results are very important and could be utilized in breeding and multiplication programs to develop new rosehip fruit and rootstock cultivars.

Effect of Explants Length and Density on in Vitro Rooting of Pineapple Ananas Comosus L Merr

Pineapple explants of different length (5, 10, 15, and 20 mm long) were cultured at different densities (one, two, three, four, and five explants per culture) on full strength, agar solidified MS medium supplemented with sucrose at 30 g/l and IBA at 2.0 mg/l for 60 days. The rooting ability (rooting %, root number, and length) of the explants were not affected by the explants' length and explants' densities per culture. Irrespective of length and densities, 82 % of the explants rooted and produced 4.1 roots each 17.7 mm long. Moreover, overall explants lengths, plantlet height was not affected by explants density and were about 50.5 mm tall. On contrary, overall density, explant of different lengths developed into plantlets of significantly different height. Plantlets' height increased from 39.2 to 49.6; 55.9 and 57.4 mm as the explants length increased from 5 to 10; 15 and 20 mm respectively. Keywords: Pineapple; Ananas comosus; Explants length; Explants density

Bio-interaction of Agrobacterium rhizogenes with Capsicum annuum L. (sweet variety) and Establishment Hairy Roots Cultures

Genetic transformation using Agrobacterium is one of the techniques used to transfer desired genes to plants. This protocol is considered a short – cut to get transformed plants which could be an alternative method and suitable system compared with the classical methods. This study aimed to investigate the response of C. annuum seedlings (sweet variety) to the formation transformed hairy roots induced by A. rhizogenes strain R1601. Sterilized seeds of Capsicum annuum were inoculated with the Agrobacterium rhizogenes inoculum. The samples were then transferred to the surface of solidified MS medium. Hairy roots were developed at the inoculation sites and were enucleated 1.0-1.5 cm length and placed in 9.0 cm Petri-dishes containing 15 ml of agar solidified MS medium. Agropine test was performed according to the standard method. The inoculated seedlings showed a good response 90%. Transformed hairy roots were established at the injection sites within 10 days and these roots were easily grown on agar-solidified MS medium. The results are confirmed that these roots were transformed roots it in terms of positive agropine detection. The current study concluded that the biological interaction between Agrobacterium rhizogenes strain R1601 and Capsicum anuum L. seedlings, was successful. This study encourages future research to improve this plant by continuing and applying modern technologies to obtain genetically modified plants.

Bio-interaction of Agrobacterium rhizogenes with Capsicum annuum L. (sweet variety) and Establishment Hairy Roots Cultures

Genetic transformation using Agrobacterium is one of the techniques used to transfer desired genes to plants. This protocol is considered a short – cut to get transformed plants which could be an alternative method and suitable system compared with the classical methods. This study aimed to investigate the response of C. annuum seedlings (sweet variety) to the formation transformed hairy roots induced by A. rhizogenes strain R1601. Sterilized seeds of Capsicum annuum were inoculated with the Agrobacterium rhizogenes inoculum. The samples were then transferred to the surface of solidified MS medium. Hairy roots were developed at the inoculation sites and were enucleated 1.0-1.5 cm length and placed in 9.0 cm Petri-dishes containing 15 ml of agar solidified MS medium. Agropine test was performed according to the standard method. The inoculated seedlings showed a good response 90%. Transformed hairy roots were established at the injection sites within 10 days and these roots were easily grown on agar-solidified MS medium. The results are confirmed that these roots were transformed roots it in terms of positive agropine detection. The current study concluded that the biological interaction between Agrobacterium rhizogenes strain R1601 and Capsicum anuum L. seedlings, was successful. This study encourages future research to improve this plant by continuing and applying modern technologies to obtain genetically modified plants.

Morphophysiological responses of Aechmea blanchetiana (Bromeliaceae) to excess copper during in vitro culture

The use of plants as bioindicators can be effective for environmental monitoring, mainly related to heavy metals such as copper (Cu). In vitro conditions are more easily controlled, to enable isolating the stress factors that can interfere in plants’ responses. The aim was to investigate the morphophysiological responses of Aechmea blanchetiana submitted to excess Cu, and to verify the degree of tolerance and potential as a bioindicator of this species. Previously cultured shoots of A. blanchetiana were transferred to agar-solidified MS medium. After 60 days, the tank system was recreated, with the addition of stationary liquid MS medium with different concentrations of Cu (0, 25, 50 and 100 μM). After 90 days with excess Cu, anatomical analysis along with quantification of the contents of nutrients, photosynthetic pigments and chlorophyll a fluorescence were carried out. Alterations in the stomatal density and chlorenchyma thickness were observed in the plants grown with excess Cu. There was an increase in the Cu content in the plants in function of the Cu levels. However, the adjustments in the content of the other nutrients were essential for tolerance to excess Cu. Aechmea blanchetiana tolerates high Cu concentrations and has potential for use as a bioindicator.

An Efficient Agrobacterium-Mediated Genetic Transformation Using Embryonic Axis in Cotton (Gossypium hirsutum L.)

Agrobacterium-mediated genetic transformation approach allows for introducing novel genes in cotton (Gossypium hirsutum L.). Development of efficient regeneration and transformation protocol is very important for recalcitrant plants like cotton. In the present study, five-day-old germinated mature embryo parts especially embryonic axis, hypocotyl and plumule of cotton ‘Lashata’ were wounded and inoculated with A. tumefaciens strain GV2260 harbouring plasmid p35S-GUS-INT. The binary plasmid p35S-GUS-INT contains neomycin phosphotransferase II (NPTII) gene driven by nopaline synthase (NOS) promoter and β-glucuronidase (GUS) gene controlled by cauliflower mosaic virus (CaMV) 35S promoter. After inoculation, explants were co-cultivated in liquid and agar-solidified MS medium for 48 h in dark condition. Agar-solidified co-cultivation medium increased transformation frequency as compared to liquid medium. The putative primary transformants were confirmed with histochemical GUS assay, PCR and RT-PCR. However, comparing the three culture explant, the embryonic axis explants had significant difference with embryonic hypocotyl and plumule explants in terms of number and percentage (%) of GUS, PCR and RT-PCR positive plant. The total transformation efficiency was recorded as 3% with varying GUS expression levels.

Response of some durum wheat (Triticum durum Desf.) genotypes on tissue culture parameters

Bu araştırmada Türkiye makarnalık buğday tarımında öneme sahip ve biyotik-abiyotik stres faktörlerine yüksek adaptasyon özelliklerinden dolayı ıslah programlarında ebeveyn olarak yararlanılan 13 makarnalık buğday genotipi (Altın 40/98, Ankara 98, Berkmen-469, Çeşit 1252, Eminbey, İmren, Kızıltan-91, Kunduru 414/44, Kunduru 1149, Meram 2002, Mirzabey 2000, Selçuklu 97, Yelken 2000) kullanılmıştır. Bu genotiplerin, kallus kültürü parametrelerine olan tepkileri belirlenerek biyoteknolojik karakterizasyonları yapılmıştır. Olgun embriyoların kullanıldığı araştırmada, steril koşullarda tohumlardan çıkartılan embriyolar 2 mg l-1 2,4-D ve MS mineral maddelerini içeren katı besin ortamında kültüre alınmıştır. Gelişen kalluslar, sürgün ve kök oluşturabilmeleri için hormon içermeyen MS ortamına transfer edilmiştir. 13 makarnalık buğday genotipinin olgun embriyoları karşılaştırıldığında; kallus ağırlığı, rejenerasyon kapasitesi ve kültür etkisi parametrelerinde genotipler arasındaki farkın P<0.01 düzeyinde önemli olduğu belirlenmiştir. Test edilen 13 makarnalık buğday genotipinden Berkmen-469, Kızıltan-91 ve Eminbey en yüksek rejenerasyon kapasitesi ve kültür etkisi değerlerinin oluşturarak, rejenerasyon yeteneği en yüksek kallusları meydana getirmişlerdir. Bu genotiplerin doğrudan gen aktarılabilme potansiyellerinin diğer genotiplere göre daha yüksek olduğu ve önümüzdeki dönemlerde biyoteknolojik yöntemlerle dayanıklı çeşit geliştirilmesine taban oluşturabilecek genotipler olduğu belirlenmiştir.

Breaking of seed dormancy in Iris suaveolens Boiss. et Reuter under in vitro conditions

Iris species have great economic importance for their use in ornamental and pharmaceutical industry. Flora of Turkey reports about 43 local taxons of which 16 are endemic. Iris suaveolens Boiss. et Reuter is an endemic species with high seed dormancy and has high tolerance to cold and drought. This study focused on seed dormancy break of I. suaveolens under in vitro conditions. In the first experiment, the seeds were given stratification treatment on MS medium containing different concentrations of BAP with or without NAA. The 2nd experiment reports effects of alternative combinations of cold (at 4°C, 24 h dark) – warm (16 h light photoperiod) conditions on medium containing different concentrations of plant growth regulators on agar solidified MS medium. Both experiments showed about 8.33% seed germination against 64.5% seed viability as confirmed by tetrazolium test. All germinating seeds were abnormal and very weak. In the third experiment, 10 minutes acid scarified seeds cultured at 24°C in 16 h light photo period on MS medium achieved 60.0% germination. Similar treatment under cold + dark condition was inhibitory and failed to show identical results. This underlines the importance of acid scarification, photoperiod and warm treatments of I. suaveolens seeds to break seed dormancy. All germinated seeds showed normal growth and development under field conditions.

Potential of tragacanth gum as gelling material in plant tissue culture studies

The diffusion ability and rheological properties of MS medium for germination and growth of tobacco seedlings of cv. Samsun Canik at different pH and concentrations of tragacanth gum were compared. The results showed that 11 g/l tragacanth gum with low osmotic pressure at pH 5.6 - 5.8 offered the best conditions for morphological developments like longer shoots, roots and broader leaves of the seedlings compared to the similar morphological developments on agar solidified MS medium with high osmotic pressure. No aberration was noted in nu1mber of chromosomes of germinated tobacco seedlings on either agar or tragacanth containing medium. On the basis of present findings, it is possible to suggest that tragacanth gum had high potential to replace agar in seed germination studies.

Plant regeneration of Nerium oleander L. from alginate-encapsulated shoot explants after short-term cold storage

ABSTRACT In this study, an efficient protocol for the regeneration of encapsulated explants of oleander (Nerium oleander L.) has been developed. Shoot tips and 1st nodal segments below the shoot tip, from in vitro-derived oleander microshoots, were encapsulated in 2.5% sodium alginate prepared in liquid MS sucrose-free nutrient medium and hardened in 50 mM of calcium chloride producing solid beads, uniform in shape. These artificial seeds, irrespective of their maintenance under light or in darkness, germinated at frequencies of 38.8–42.2%, producing 3.0–3.3 microshoots per bead. In the case of using 100 mM of calcium chloride for hardening, the beads were firm, of uniform globular shape and suitable for handling, exhibiting a germination response of 68.9%. Encapsulated shoot tip explants, following storage at 4°C for 8 weeks, exhibited a higher regeneration response (60.0%) than non-encapsulated similar explants stored under the same conditions (11.1%). Microshoots, excised from cold-stored encapsulated explants after germination, rooted easily in agar-solidified MS medium with 2 μΜ IBA and after their transplantation into a peat-perlite substrate (3:1, v/v), were acclimatised successfully and established in the greenhouse with minimal losses. The present encapsulation procedure could be applied as an alternative method of micropropagation of desirable elite clones of oleander.

EFFECT OF BA AND TDZ IN VEGETATIVE MULTIPLICATION OF SHOOT TIPS AND NODES OF Buahinia purpurea L. In vitro

This study aimed to develop a system for propagating of Camel's Foot Bauhinia purpurea L. by tissue culture technology. Seeds were germinated after removal the external coats in agar-solidified MS medium without growth regulators, In order to get fresh explants and sterilized for use in the following study stages. After 4 weeks of germination and a suitable high of seedlings explants from growing tips and nodes were excised and cultured in MS medium supplemented with different concentrations of TDZ and BA (0.0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg/l) of each during initiation and replication stages in order to get a highest number of multiplied new shoots, Cultured growing tips showed that 1.5 mg/l of BA produced the shoots numerous average 2.66 using 2.5 and 3.0 mg/l of TDZ also have largest shoots average 3.10. On the other hand, nodes cultured on 3.0 mg/l BA have highest shoots average 6.44 at a time TDZ with concentration of 2.0 mg/l also have highest shoots number 7.44. Results showed in multiplication stage for growing tips and nodes which we have from initiation stage cultured in solid MS medium with same concentration of BA and TDZ have a good responses. So BA with 1.5 mg/l of concentration has largest shoots numbers 7.55 when 2.5 mg/l BA was the best in case of shoots length with 33.94 cm. But culturing nodes showed that 3.0 mg/l of BA was the best and have largest averages of shoot numbers 8.22 when in the case of shoots numbers characteristics we found that the concentration of 2.0 mg/l BA was the best with record of 21.49 cm. The results proved that TDZ in 2.0 mg/l have largest average shoots numbers 9.66.

Effect of Explants Length and Segmentation and Separated and Cluster of Shoots on the in vitro Shoot Formation and Growth of Pineapple (Ananas comosus L. Merr.)

Two experiments were conducted. In one, multiple shoots complex from Smooth cayenne pineapple stock cultures were cut into cluster of two, three, four shoots and were also separated into individual shoots of different length 5, 10, 15, 20 and 25 mm long and cultured on agar solidified MS medium enriched with benzylaminopurine (BAP) at 2.23 mg/l. In the other one, 25 mm long shoots were cut longitudinally into halves and quarters and cultured at density of one explant and 15 mm long shoots were cultured intact at density of one, two, three and four explants per culture. Longer (25 mm) intact and halved explants produced the highest (10 shoots) while 5 and 10 mm long explants resulted in lowest (6 shoots) and 15 and 20 mm long explants resulted in an intermediate (8 shoots) shoot formation. Shoot formation whether the explants were cultured as separated intact or as cluster of shoots decreased from 10 to 4 shoots per explant as the density increased from one to four. Shoot length was not affected by using of separated intact explants of different length. However, increasing the explants density increased the shoots length particularly when cluster rather than separated shoot were used as explants.

Uzun Gün Şartlarında Patates (Solanum tuberosum L.)’in Mikroçoğaltımında Jasmonik Asidin Etkisi

Meristem derived in vitro plantlets of potato ( Solanum tuberosum L.) cultivars Pasinler, Granola, and Caspar were micropropagated on agar-solidified MS medium containing different concentrations of jasmonic acid (JA)using single-node stem cuttings. The results of the study showed that inclusion of 1.0 µM JA in the MS medium provided a substantial and visible increase in most of the plantlet characteristics studied and the effect of this concentration was more pronounced compared to the control and all other JA concentrations. The highest number of shoots (8.00), nodes (19.00), leaves (19.00), and roots (15.75) on cv. Caspar and a maximum shoot length (15.13 cm) was observed on cv. Granola from 1.0 µM JA concentration. This concentration of JA also helped to induce longest roots (14.95 cm) on cv. Pasinler. The highest plantlet fresh weight from cv. Pasinler (649.38 mg), and cv. Caspar (630.18 mg), and maximum dry weight of plantlets from cv. Pasinler (98.58 mg), cv. Caspar (93.89 mg) and cv. Granola (79.03 mg) were noted on MS medium containing 1.0 µM of JA. Since the application of JA accelerates the multiplication of young plantlets throughout the year without depending on the season, this allows fast commercial propagation of new potato cultivars. These results may also serve as basis for the mass production of these economically important potato cultivars through in vitro micropropagation techniques and microtuberization studies

Somatic embryogenesis and plant regeneration from cotyledonary node's calli of Trigonella foenum-graecum L.

The present study describes plant regeneration via somatic embryogenesis from cotyledonary node callus. Callus cultures were derived from cotyledon node segment excised from Trigonella foenum-graecum L. axenic seedlings grown in agar solidified Murashing and Skoog's(MS) medium supplemented with Benzyl adenine BA at 2.0 mg l-1 and Naphthalene acetic acid NAA at 1.0 mg l-1. These cultures were stimulated to form somatic embryoids at 32% frequency of in agar-solidified MS medium containing NAA at 0.5mg l-1 in the presence of 15% v/v coconut water. Light microscopy examination of sections of heart embryos stage proved the presence of vascular system. Transfer of other developmental stages of embryos to the same induction medium led to the development of secondary somatic embryos and embryoids into plantlets in agar-solidified MS medium lacking hormones. They were readily rooted in MSO and successfully transferred to soil.

Regeneration of Cucumis melo L. plants from cell suspension derived from hypocotyls callus

This study aimed to investigate the regeneration capability of cell suspension derived from hypocotyl callus of the vegetable plant, muskmelon, Cucumis melo L. Culture of the densities (7.5, 9.8, 13.0, 11.2, 7.8, 4.2 )× 102 cell/ml produced callus primordia which formed typical callus culture successfully. This green – yellowish color and semi-compact callus regenerated shoots on agar solidified MS medium supplemented with 2.0mgL-1BA. These regenerates rooted readily in MS0 medium. They were adapted and routinely transferred to soil. The conclusion of this work that muskmelon plants have a good response to in vitro culture with no need to specific requirements.

Callus Cultures derived from electrotreated cell suspension of Dianthus caryophyllus L. embedded in agar drops express their totipotency

Callus cultures were produced from leaf, excised from axenic carnation, Dianthus caryophyllus L. seedling, on agar-solidified MS medium supplemented with 0.5 mgl-1 of 2,4-D and 0.1 mgl-1 of BA. Cell suspension was initiated in liquid MS medium containing the above growth regulators. This suspension reported the density of 143 × 103 cell /ml and its viability was ranged between 41-90%. Samples of cell suspension represents seven different densities were exposed to three electrotreatment 300v/50 msec, 500v/100msec. and 700v/150 msec. These electrotreated samples were cultured by embedding in to agar drops in solid- liquid cultures. Callus derived from cell suspension expressed their totipotency forming carnation plants.