Shoot Tip Explants Research Articles

Direct regeneration of plantlets from shoot tip explants of a vulnerable medicinal plant Celastrus paniculatus Willd.

Direct regeneration of plantlets from shoot tip explants of a vulnerable medicinal plant Celastrus paniculatus Willd.

IN VITRO STORAGE OF PAULOWNIA TOMENTOSA

Storage of plant species with high commercial potential as ornamental or for the alimentation and pharmacological has been used to preserve genetic variability and allowed the study of its properties in a controlled environment through the use of in vitro culture techniques. In this study, shoot tip explants of Paulownia tomentosa were excised from in vitro grown shootlets and cultured on Murashige and Skoog (MS) medium supplemented with 30 g l-1 sucrose (control) and different concentrations of sucrose or sorbitol (20, 40, 50 and 60 g l-1) and in combinations (10 + 10, 20 + 20, 25 + 25 and 30 + 30 g l-1) and incubated under 18 °C in complete darkness for 2, 4, 6, 8 and 10 months. Results showed that all shoot tip explants conserved on MS medium augmented with different concentrations of sucrose or sorbitol and their combinations for 2 months, at the level of 30 g l-1 sucrose (control) for 4 months recorded the best survival percentage (100%). At the end of storage period (after 10 months), the highest survival percentage (83.33%) was noticed when explants were conserved on MS medium with 30 g l-1 (control) or 50 g l-1 sucrose. Meanwhile, the lowest survival percentage (25.00%) was achieved when explants were conserved on MS medium with 10 g l-1 sucrose + 10 g l-1 sorbitol. Moreover, it was observed an increasing of shoot length and number of proliferated shoots/explant during storage periods except all treatments with sorbitol and the treatment with sucrose plus sorbitol (10 + 10 g 1-1) remained without change in all storage periods.

In vitro propagation of Satureja thymbra L. (Lamiaceae): A valuable aromatic-medicinal native plant of the Mediterranean region

Satureja thymbra L. or savory is a highly threatened aromatic-medicinal endemic Mediterranean plant. Micropropagation can play a role in the protection of the natural eco­system. The disinfection protocol involved the immersion of shoot-tip explants in 70% alcohol (1 min) and 3% NaOCl (15 min) giving 61.18% success. The MS medium supplemented with 30 g/l sucrose and 7 g/l Plant Agar was optimum for the initial establishment stage (10-day culture, 92.31% proliferation) and afterwards supplemented extra with 0.5 mg/l BA for multiple shoot induction (5 weeks) (100% proliferation, 5.5 shoots 14.24 mm long). In rooting stage, 1 mg/l IBA gave the highest rooting (58.62%), 0.5 mg/l NAA the maximum root number (12.13) ​​and 0.1 mg/l IAA the largest root length (34.35 mm) (MS medium + 20 g/l sucrose + 6.5 g/l Plant Agar, 7 weeks) (Experiment No. 1). There was a progressive increase in rooting and shoot proliferation increasing the number of subcultures, being highest (100% rooting, 6 roots 70 mm long, 38.1% shoot multiplication, 2 shoots 30 mm long) in the 4th subculture/ 4.5 weeks each in WPM medium (Experiment No. 2). Therefore, best rooting results (50-100%) were accomplished in 3 different composition media: (1 and 2) WPM medium + 20 g/l sucrose + 6 g/l Plant Agar (1st and 4th subculture) and (3) MS medium + 30 g/l sucrose + 1 mg/l IBA + 7 g/l Plant Agar. The ex vitro survival of rooted microshoots after 4 weeks in the greenhouse mist (1 peat moss: 1 perlite) was 81.82%.

A simple and rapid protocol for the genetic transformation of Ensete ventricosum

Enset (Ensete ventricosum), also known as Ethiopian banana, is a food security crop for more than 20 million people in Ethiopia. As conventional breeding of enset is very challenging, genetic engineering is an alternative option to introduce important traits such as enhanced disease resistance and nutritional value. Genetic transformation and subsequent regeneration of transgenic enset has never been reported mainly due to challenges in developing transformation protocols for this tropical species. Agrobacterium-mediated transformation could be a practical tool for the genetic improvement of enset. However, the efficiency of the transformation system depends on several parameters such as plant regeneration, genotype, explant, selection agent and Agrobacterium strains. As a first step towards the development of transgenic enset, a simple and rapid plant regeneration system was developed using multiple buds as explants. Induction and proliferation of multiple buds from shoot tip explants was achieved on Murashige and Skoog (MS) medium supplemented with 5 and 10 mg/l of 6-benzylaminopurine (BAP), respectively. Shoots were regenerated from multiple buds on MS media containing 2 mg/l BAP and 0.2% activated charcoal. Based on the optimized regeneration protocol, an Agrobacterium-mediated transformation method was developed using multiple buds as explants and the binary plasmid pCAMBIA2300-GFP containing the green florescent protein (gfp) reporter gene and neomycin phosphotransferase II (nptII) selection marker gene. Transgenic plantlets were obtained within 4 months at a frequency of about 1.25%. The transgenic lines were validated by PCR analysis using primers specific to the nptII gene. To obtain uniformly transformed plantlets, chimerism was diluted by subculturing and regenerating the transgenic shoots on a selective medium containing kanamycin (150 mg/l) for five cycles. The uniformity of the transgenic plants was confirmed by Southern blot hybridization and RT-PCR analyses on different tissues such as leaf, pseudostem and root of same transgenic plant. In the present study, we report a simple Agrobacterium-mediated transformation system for generating transgenic events of enset. To the best of our knowledge, this is the first report on the stable transformation and regeneration of transgenic events of enset. The transformation system established in this study can be used for the generation of transgenic enset with important traits such as disease resistance.

The effectiveness of light emitting diodes on shoot regeneration in vitro from shoot tip tissues of Limnophila aromatica (Lamk.) Merr. and Rotala rotundifolia (Buch-Ham. ex Roxb) Koehne

ABSTRACTLight emitting diodes (LEDs) have become a promising technology for agriculture and horticulture. I investigated the effects of white (W), red (R) and blue (B) LED lights on the propagation of Limnophila aromatica (Lamk.) Merr. and Rotala rotundifolia (Buch-Ham. ex Roxb) Koehne using tissue culture. The shoot tip explants of L. aromatica and R. rotundifolia under different light environments were cultured in Murashige and Skoog (MS) basic nutrient medium with 6-benzylaminopurine (BAP) (0.05, 0.10 and 0.20 mg/l) and gibberellic acid (GA3) 0.25 mg/l) + kinetin (KIN) 0.25, 0.50 and 0.75 mg/l. The explants grown under combinations of white, red and blue LEDs were more effective for propagation of the plants in vitro. In L. aromatica, the maximum number of shoots/explant and the longest shoot lengths were obtained using the combination of white, red and blue LED lights in a 1:2:1 ratio in MS medium supplemented with 0.10 and 0.20 mg/l BAP. In R. rotundifolia, the maximum shoots/explant and shoot lengths were obtained in the explants using the combination of white, red and blue LED lights in a 1:2:1 ratio in the MS culture media fortified with 0.25 mg/l GA3 + 0.25 and 0.75 mg/l KIN. After the regenerated shoots were rooted, they were adapted successfully to external conditions. LEDs have significant advantages over fluorescent lights.

In vitro Multiple Shoot Regeneration from Petunia hybrida

An efficient plant regeneration protocol was developed from in vitro germinated seeds of Petunia hybrida an ornamentally important plant in the family Solanaceae. Shoot tip and node explants of Petunia hybrida were cultured on MS basal medium supplemented with different concentrations and combinations of Benzyl amino purine (BAP), 1-Naphthaleneacetic acid (NAA), Indole-3-butyric acid (IBA) and Gibberellic acid (GA3). The highest shoot length was obtained from MS medium supplemented with 1 mg/l BAP + 1 mg/l NAA. The highest shoot number (3 shoots/explant) were obtained from MS medium supplemented with 0.6 mg/l BAP + 0.5 mg/l IBA. The isolated shoots were transferred to MS basal medium supplemented with different concentrations of GA3 ranging from 0.05, 0.2, 0.5 and 1 mg/l for shoot elongation. The highest shoot length (5.75 cm) was recorded from the MS medium supplemented with 0.2 mg/l GA3 +0.2 mg/l BAP. Rooting of regenerated shoots were achieved on MS medium supplemented with 0.1-1 mg/1 IBA and NAA. The regenerated shoots with well developed roots were successfully acclimatized and established in pots containing sterilized peat moss and grown under laboratory conditions with 70% survival rates.

The Efficient Germination Method For Marhmallows With Medicinal Importance: The Nicking on Seed Coat

Malvaceae familyasının üyelerinden biri olan Alcea cinsi tıbbi öneme sahip bazı türleri içermektedir. Alcea kurdica Alef. (Devegülü Hatmi) tonsilite, gastrik ülsere, zatürreye, boşaltım sistemi enfeksiyonlarına ve saç kırana karşı kullanılan tıbbi bir bitkidir. Bu çalışmanın amacı, devegülü hatmi bitkisi için steril şartlar altında etkili bir çimlendirme yönteminin geliştirilmesi ve bu bitkinin doku kültürü çalışmalarında kullanılabilir hale getirilmesidir. Bu amaç için, steril bir ajan olan sodyum hipokloritin üç farklı konsantrasyonu (%5, %7.5 ve %10), 42°C sıcaklıkta inkübasyonu ve tohum kabuğunun bistüri ile çentiklenmesi uygulamaları test edilmiştir. %5 ve %7.5 sodyum hipoklorit konsantrasyonlarının sterilizasyon için yeterli olmadığı belirlenirken, %10 sodyum hipoklorit konsantrasyonun ise yeterli olduğu gözlenmiştir. Çimlenme indeksi sonuçlarına göre, kabuğu çentiklenen tohumlar hızlıca çimlenirken, 42°C de inkübasyonun çimlenmeyi etkilemediği gözlenmiştir. Yedinci günde, kotiledon, kotiledon sapı ve hipokotil dokuları doku kültürü için kullanılabilir duruma gelmiştir. Sekizinci günde, yapraklar oluşmaya başlamıştır. Yaprak, yaprak sapı ve sürgün ucu eksplantları ise otuzuncu günde doku kültüründe kullanılabilecek duruma gelmiştir. Sonuç olarak, bu çalışma ile tıbbi öneme sahip devegülü hatmi için etkili bir çimlendirme yöntemi geliştirilmiştir.

In vitro shoot multiplication of Rumex vesicarius L., and quantification of ascorbic acid and major phenolics from its leaf derived callus.

An optimised method for enhanced in vitro shoot multiplication of Rumex vesicarius (Polygonaceae)-a branched succulent herb-was achieved. The in vitro seed pre-treatment with 4% urea was able to show 95% seed germination on MS medium within 2weeks of culturing. In vitro shoot bud induction from shoot tip explants was best in the presence of 2mgL-1 kinetin on Murashige and Skoog medium (MS medium), wherein 8.3 shoots/explants were observed. Shoot elongation was found to be high (3.75 ± 0.5cm) in 2mgL-1 BA comprising medium. The transfer of micro-shoots on to MS medium comprising 1.5mgL-1 IBA and 1% activated charcoal (w/v) supported efficiently in vitro rooting (2.5-4.0cm) in 3-4weeks. Upon hardening, 70% rooted plants survived under greenhouse conditions. Though friable callus was produced on MS medium-containing 2mgL-1 BA followed by 3mgL-1 BA, no organogenesis was noticed. The ascorbic acid content of 78.62 ± 0.25mg 100g-1 FW was recorded in callus cultures grown on medium supplemented with 2mgL-1 BA, and it is 1.74-fold more compared to normal ex vitro leaves of the same age. In vitro raised plant leaf showed 1.98-fold more ascorbic acid (89.42 ± 0.18mg100g-1 FW) to that of ex vitro leaves. The total phenolic content was found to be 60mg in callus as compared to 610mg (per 100g GAE FW) of ex vitro leaves. The major phenolic compounds quantified were synergic, chlorogenic, ferulic, and generic acids, respectively. This optimised protocol will facilitate to pursue scale-up studies for in vitro ascorbic acid production and also to further investigate the kinetics of biosynthetic pathway genes involved.

Clonal Propagation In Vitro of Paphiopedilum Hybrids from Adult Plants

The aim of this study was to develop an efficient protocol for shoot tip culture from adult plants of Paphiopedilum Pfitzer. A considerable seasonal effect on explant collection was observed in the aseptic cultures established from adult plants, including the survival and microbial contamination of explants. The shoot tip explants excised from adult plants in February and May showed higher survival and had less contamination than those explants excised in August and November. Moreover, the season of explant collection also affected the subsequent shoot forming capacity and multiplication of axillary buds. In Paphiopedilum ‘In-Charm Silver Bell’, higher shoot forming capacity was observed in February and May, whereas higher shoot multiplication was observed only in February. In Paphiopedilum ‘Hsinying Maudiae Leopard’, both February and May were optimal timing for shoot forming capacity and multiplication. We also demonstrated the effectiveness of transcinnamic acid (tCA), an antiauxin chemical in diminishing the apical dominance of shoot tip explant and thus improving the axillary bud outgrowth. In P. ‘In-Charm Silver Bell’, the addition of 100 μM tCA plus 13.3 μM 6-benzylaminopurine (BA) for 1 month promoted axillary shoot bud formation from shoot tip explants as compared with the control.

In vitro propagation of Codonopsis javania (Blume) Hook. f. et Thomson through the callus induction

Codonopsis javania (Blume) Hook.f. et Thomson a traditional medicine plant and now an endangered species in Vietnam is grown for roots. The research was carried out to establish the plant propagation for the purpose of concerving and exploting this endangered medicinal herbs. In vitro shoot tip explants (1 – 1.5 cm) were induced to form callus on MS medium containing NAA (0.5 – 2 mg /L) with TDZ 0.1 mg/L. After four weeks of culture, in the MS medium combine with NAA 1 mg/L and TDZ 0.1 mg/L the explant induced compact callus (green, solid) wsa achieved 85.33%. The callus induction to form shoots on medium MS containing BA (0.5 – 2.0 mg/L) with NAA 0.2 mg/L. After 4 weeks of culture, shoot formation was higher in the MS medium containing BA 1.0 mg /L and NAA 0.2 mg/L and achieved of 82.67 % with 9.92 shoots/explant. The best shoot proliferation (2 – 3 cm) was excised and transferred to a medium shoot multiplication with the same composition as the shoot induction medium in which NAA 0.2 mg/L was replaced by NAA 0.5 mg/L. When compared the shoot multiplication between the two mediums at the same BA concentration (2 mg/L), all shoots increased and reached 5.87 times after 60 days cultured. On rooting MS medium with IBA 1 mg/L, 88.67 % in vitro rooting was observed with the average root yield of 4.33 roots/shoot and the length of 8.27 cm. Root length and their yield quality were highly improved when using of coconut fiber (30 %) and earthworms compost (70 %) (v/v) in the transfer medium after acclimatisation stages.

Effect of The Ethylene Inhibitor “Agno3”, Vitamin B9 “Folic Acid” And Thiol Compound “GSH” On in Vitro Propagation of Sideritis Syriaca L. Subsp. Syriaca (Hellenic Mountain Tea of The Crete Island)

The aim of this research was to study the effect of the ethylene inhibitor “silver nitrate (AgNO3)” and vitamin B9 "folic acid" in different concentrations combined with cytokinin BA as well as the antioxidant thiol compound “L-glutathione reduced (GSH)” in different concentrations simultaneously with auxins (IBA+NAA) on micropropagation efficiency of the endemic Sideritis syriaca L. subsp. syriaca (Hellenic mountain tea of Crete/ Malotira) using shoot tip explants. The culture medium used was the MS supplemented with 30 g/l sucrose. The simultaneous application 5 ?M AgNO3 + 2.2 ?M BA promoted best the initial shoot induction stage exhibiting 4.5 shoots/ explant and 100% shoot multiplication (5 weeks). Folic acid applied at 0.25 mg/l in combination with 0.5 mg/l BA exhibited the highest shoot multiplication percentage (90%) (4 weeks). GSH at 10 ?M with 10.7 ?M NAA + 4.92 ?M IBA gave the greatest root length (13.68 mm), at 25 ?M caused a 3-fold increase in rooting (90%) and 250 ?M GSH raised by 20% shoot multiplication (80%). An 89% final survival rate of rooted microplants to ex vitro unheated greenhouse conditions was recorded within 8 weeks period during mid-late spring. Thus, the acclimatization and hardening process was successfully completed.

MORFOGENESIS ANGGREK (Anoectochilus formosanus) SECARA IN VITRO

ABSTRACT Anoectochilus formosanus is one species of Orchids, known as a “Jewel Orchids” and have been used as a folk medicine in China and Taiwan.The aim of this study was to examine the response of using different types of explants and combination of growth regulators TDZ and BAP to morphogenesis of Anoectochilus formosanus Orchids by in vitro. The results showed that using different types of explants had very significant effect to the percentage of callus and shoot morphogenesis on 12 weeks after planting, the number of adventitious buds, adventitious shoot length, and callus diameter on three weeks until 12 weeks after planting. The best explant to callus morphogenesis was showing on shoot tip explant (50%) and to shoot morphogenesis was showing on auxillary buds and internode explant (100%). The best number of adventitious buds was observed in second internode explant with average number of shoots are 7.62 shoots. The best adventititous length was observed in axillary buds explant with average number of shoot length in 1.16 cm, and the best callus diameter was observed in shoot tip explant with average of diameter in 2.2 mm. The combination of plant growth regulator TDZ and BAP had a very significant effect to adventitious length on 10 weeks after planting, and a significant effect to adventitious length on 11 and 12 weeks after planting. The best adventitious length was observed on 0.25 mg L -1 TDZ + 0.75 mg L -1 BAP in 1.13 cm per explant on 12 weeks after planting. The best combination to callus and shoot morphogenesis was observed on 0.75 mg L -1 TDZ + 0.25 mg L -1 BAP. There was no interaction between used a different types of explant and combination plant growth regulator to morphogenesis Anoectochilus formosanus orchids by in vitro.

Micropropogation of Sideritis pisidica Boiss. et Heldr. Apud Bentham

Sideritis pisidica Boiss. et Heldr. Apud Bentham is known as a medicinal and aromatic plant. Sideritis pisidica Boiss. et Heldr. Apud Bentham is a plant which is endemic to Turkey and a threatened species in the Labiateae family. This species is used in folk medicine since ancient times. Micropropogation is an alternative method for rapid clonal propogation. In this study, Sideritis pisidica Boiss. et Heldr. Apud Bentham was micropropagated using MS media containing different combinations of plant growth regulators; BA (0, 0.5, 1 and 1.5 mg L‑1) and GA3 (0, 0.5, 1 and 1.5 mg L‑1) for propagation, IBA (indole-3-butyric acid) 0, 0.5, 1, 1.5 and 2 mg L‑1) for rooting. The MS medium supplemented with 0.5 mg L‑1 BA and 0.2 mg L‑1 GA3 (giberellic acid) showed highest percentage of shoot multiplication and rooted with 85.5 and 79.5% response from shoot tip explant, respectively.

In vitro Mass Propagation of Cymbidium aloifolium (L.) Sw.

Cymbidium aloifolium (L.) Sw. is an epiphytic orchid distributed locally in Bangladesh. Natural populations of this orchid are under threat due to its high commercial value in the floriculture industry by over exploitation. Mass propagation provides an alternative means of satisfying the demand. In this study, half strength of MS supplemented with BAP or different combinations with NAA was evaluated for seed germination and early protocorm development of C. aloifolium (L.) Sw. The maximum percentage of seed germination (98) was obtained on modified MS (half strength of micro and macro nutrients and full strength of vitamin) supplemented with 0.5 mg/l BAP and 0.5 mg/l NAA after three weeks of culture. Maximum number of shoots (9.6 shoots per single shoot) were developed from shoot tip explants on half strength of MS supplemented with 1.0 mg/l BAP and 0.5 mg/l NAA. The most effective auxin source promoting root production (3.2 roots per shoot) was 1.0 mg/l IBA in combination with 0.5 mg/l BAP. The seedlings were successfully acclimatized (survival percentage 99) in a nethouse using a rooting medium of crushed sterile brick, cocopit and charcoal (1 : 1 : 1 v/v).
 Plant Tissue Cult. & Biotech. 29(1): 73-79, 2019 (June)

The Stimulating Effects of Imazalil and Carbendazim Fungicides on in Vitro Propagation and ex Situ conservation of the Medicinal Balkan Range-Restricted Sideritis Raeseri Boiss & Heldr. Subsp. Raeseri

Sideritis raeseri Boiss & Heldr. subsp. raeseri, known in Greece as Mountain tea of Parnassus or Velouchi is a range restricted medicinal plant of the Balkan peninsula. Conventional propagation methods did not allow the mass production of plant material in a short period of time due to both low seed germination rate and rooting of cuttings. Therefore, the aim of this study was to establish a reliable, reproducible and efficient regeneration protocol for mass and large-scale micropropagation, germplasm and ex situ conservation of S. raeseri Boiss & Heldr. using Imazalil and Carbendazim fungicides. After 9 weeks, 2.5-10 mg/l Imazalil stimulated root length by 1 cm but diminished root number and rooting percentage. Optimum shoot number (5.58), shoot length (24.91 mm), shoot multiplication (100%), root number (20.63) and rooting (66.67%) were recorded with 0.5 mg/l kinetin (KN) + 0.05 mg/l ?-napthaleneacetic acid (NAA) (Imazalil-free). After 4 ½ weeks, 1 mg/l Carbendazim + 0.5 mg/l 6-benzyladenine (BA) yielded 5.77 shoots/explant, 16.1 mm shoot length and 100% shoot multiplication. However, Carbendazim did not result in rooting. The ex vitro survival percentage of rooted shoot-tip explants derived from Imazalil experiment was 95%. Carbendazim proved useful in shoot proliferarion and Imazalil in root elongation of S. raeseri Boiss & Heldr. subsp. raeseri micropropagation system.

Effect of glycine betaine, polyvinylpyrrolidone and D-mannitol on micropropagation of Sideritis raeseri Boiss and Heldr. subsp. Raeseri.

Despite being protected, the number of plants that belong to Sideritis genus has decreased in the last years due to the urbanization of its natural habitat. Sideritis raeseri Boiss and Heldr. subsp. raeseri known as mountain tea of Parnassus or Velouchi is a valuable genetic resource of the Greek flora because of its special characteristics, so, the development of a micropropagation protocol is crucial to its conservation and use in breeding programs. The objectives of the study were to evaluate the effects of glycine betaine (GB), polyvinylpyrrolidone (PVP-10) and D-mannitol applied in different concentrations in combination with auxins [α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA)] on shoot proliferation and rooting of shoot-tip explants of the studied species. Better shoot proliferation results were obtained in the following two combination treatments: (1) 100 mg/l GB + 0.5 mg/l NAA (5.5 shoots/explant 2.34 cm long, 75% shoot multiplication), and (2) 2.5 g/l D-mannitol + 3 mg/l IBA (2.92 shoot/explant 83.33% shoot multiplication percentage). In relation to rooting, better results were performed only with PVP at 0.5 g/l applied simultaneously with 0.5 mg/l NAA (12 roots/rooted microcutting, 1.47 cm root length, 90% rooting). The ex vitro survival percentage of rooted microcuttings derived from all 5 experiments was ranged between 68% and 93%.

Spermidine influences enhanced micropropagation and antibacterial activity in Aerva javanica (Burm. F.) Shult.

Aerva javanica is an exotic medicinal plant widely distributed in India. It possesses numerous medicinal benefits. The present study was aimed to in vitro regenerate the explants of Aerva javanica, and to quantify the components of secondary metabolites (using high performance liquid chromatography technique, HPLC) present in the wild and micropropagated samples. A comparative analysis of the antibacterial activity between wild, in vitro regenerated leaf and callus was also performed in this study. The in vitro propagation of the shoot tip explants was carried out in Murashige and Skoog’s (MS) medium supplemented with different plant growth regulators. The results of the micropropagation showed highest rate of regeneration in lower concentration of spermidine (0.5 mg/L). Phytochemicals such as flavonoids, tannins, and phenolic compounds were identified as abundant content in wild, in vitro leaf and callus materials, they were quantified by using HPLC technique. The amount of flavonoid and phenolic compound was found to be higher in in vitro leaf, followed by callus extract and wild leaf extract. The fraction of secondary metabolites possess strong antibacterial activity against the selected human pathogens viz., Staphylococcus aureus and Proteus vulgaris. The study concluded with a first report on reproducible protocol for micro propagation and sustainable utilization of Aerva javanica in the pharmaceutical fields.

Effects of polyploidization on petal characteristics and optical properties of Impatiens walleriana (Hook.)

Impatiens walleriana (Hook.) (2n = 2x = 16) is a popular flowering species in the Balsaminaceae family. In order to investigate the effects of polyploidy induction in this species, shoot tip explants measuring 1.5 cm in length were treated with 0, 15, 30, and 60 μM oryzalin for 6, 12, 24, and 48 h in vitro. After conducting the flow cytometric analysis of the plantlets, it was revealed that 68.75% of the explants treated with 60 μM oryzalin for 24 h became tetraploid (2n = 4x = 32), indicating that this treatment is the most effective for tetraploidy induction. The tetraploid plants exhibited increased leaf area, leaf thickness, ovary width, pollen cell length, and stomatal size, as well as a greater number of chloroplasts within stomatal guard cells. Furthermore, tetraploid plants showed decreases in plant height, stomatal guard cell density, and number of flowers. Epidermal conical cells were enlarged by about 1.7 times in tetraploid flowers. Optical examination of petals revealed that reflection of light from the tetraploid petals was reduced among almost all spectra. Reflection was reduced in the spectral region between 350 and 400 nm, which may have occurred as a result of changes in size of the epidermal conical cells. Furthermore, the petals in the tetraploid flowers had more total anthocyanin content. The latter two observations may explain the deepening of color in tetraploid petals. The new features observed in tetraploid flowers may be useful for generating new traits in classical breeding programs. It is also possible that these changes may be caused by alterations in gene expression and protein activity and can thus be examined to identify the genetic factors associated with flowering time and flower morphology in tetraploid I. walleriana. Further investigations are needed to study the interactions between tetraploid I. walleriana and pollinators. The latter can be important for understanding the evolutionary relationship between pollinators and the polyploidy process in this plants. Treating I. walleriana with oryzalin can produce tetraploid plants. Compared to their diploid counterparts, the tetraploid plants have significant differences in their vegetative and reproductive characteristics. Changes in the form and color of the flowers can be attributed to the changes in the size of the conical cells on the surface of the epidermis, along with the increase in the total anthocyanin content in petals.

Sucul bir bitki olan Pogostemon erectus (Dalzell) Kuntze’un In Vitro hızlı çoğaltımı

The aim of this study is to investigate the multiple and rapid production of Pogostemon erectus (Dalzell) Kuntze by tissue culture techniques. The shoot tip and nodal explants of P. erectus were isolated and then cultured in Murashige and Skoog (1962) (MS) nutrient media containing thidiazuron (TDZ) and 2,4-Dichlorophenoxyacetic acid (2,4-D) in different combinations. The first shoot formation on the explants was observed on day 15. The number of shoots per explant ranged from 2.85 to 29.39 in the shoot tip explants and from 2.72 to 32.24 in the nodal explants. The maximum number of shoots per explant was obtained in the MS medium containing 0.20 mg/L TDZ + 0.10 mg/L 2,4-D for shoot tip explant (29.39) and in the MS medium containing 0.10 mg/L TDZ + 0.10 mg/L 2,4-D for nodal explant (32.24). The longest shoots in the shoot tip (1.76 cm) and nodal (1.64 cm) explants were determined in MS medium supplemented with 0.10 mg/L TDZ + 0.10 mg/L 2,4-D. Regenerated shoots were rooted in MS medium supplemented with 0.25 mg/L indol-3yl acetic acid (IAA), and then acclimatized to aquarium conditions.

Micropropagation and Quantification of Bioactive Compounds in Mertensia maritima (L.) Gray

The goal of this study was to establish an efficient protocol for the large-scale propagation of Mertensia maritima (L.) Gray, and evaluate the carotenoid, fatty acid, and tocopherol contents in the leaves of in vitro regenerated shoots. Surface-disinfected node and shoot tip explants were placed on semisolid Murashige and Skoog (MS) medium with 0–16 µM N6-benzyladenine (BA), kinetin, (KN), and thidiazuron (TDZ) alone, or in combination with, 1 or 2 µM α-naphthaleneacetic acid (NAA). Of the three different cytokinins employed, TDZ elicited the best results for axillary shoot proliferation. A maximum frequency of shoot initiation above 84%, with a mean of 8.9 and 4.8 shoots per node and shoot tip, respectively, was achieved on the culture medium supplemented with 4 µM TDZ. A combination of TDZ + NAA significantly increased the percentage of multiple shoot formation and number of shoots per explant. The best shoot induction response occurred on MS medium with 4 µM TDZ and 1 µM NAA. On this medium, the node (93.8%) and shoot tip (95.9%) explants produced an average of 17.7 and 8.6 shoots, respectively. The highest root induction frequency (97.4%) and number of roots per shoot (25.4), as well as the greatest root length (4.2 cm), were obtained on half-strength MS medium supplemented with 4 µM indole-3-butyric acid (IBA). The presence of six carotenoids and α-tocopherol in the leaf tissues of M. maritima was confirmed by HPLC. Gas chromatography-mass spectrometry analysis confirmed the presence of 10 fatty acids, including γ-linolenic acid and stearidonic acid in the leaf tissues of M. maritima. All-E-lutein (18.49 μg g−1 fresh weight, FW), α-tocopherol (3.82 μg g−1 FW) and α-linolenic acid (30.37%) were found to be the significant compounds in M. maritima. For the first time, a successful protocol has been established for the mass propagation of M. maritima with promising prospects for harnessing its bioactive reserves.