In vitro Gynogenic Plant Production in Allium tuncelianum (Kollman) Özhatay, Matthew & Şiraneci

Allium tuncelianum (Kollman) Özhatay, Matthew & Şiraneci forms a single-cloved edible white bulb with mild garlic (A. sativum) odour and taste. Its ability to form seeds make it suitable for genetic improvement via classical and modern approaches. A detailed study was carried out to determine the gynogenesis potential of two A. tuncelianum (AT1 and AT2) accessions. Unopened flower buds of A. tuncelianum accessions were cultured in various BDS- and MS-based induction media. A total of 17 (0.09%) gynogenic plantlets were obtained from ~20000 flower buds used in gynogenesis induction experiment. Accessions showed slight differences in their responses to gynogenesis induction cultures. The highest gynogenic plantlet production frequency (0.34 %) in AT1 was achieved flower buds cultured in T12 medium (MS with 100 g/L sucrose, 1 mg/L a-naphthalene acetic acid (NAA) and 8 mg/L isopentenyl adenine (2IP). Flower buds of AT2 showed the highest gynogenic plantlet production response (0.44 %) in T2 medium (BDS with 50 g/L sucrose). Eight of 17 gynogenic plantlets continued to grow and became healthy plants with green leaves and well established roots. Flow cytometric (FCM) analysis of well-developed gynogenic plants showed that two were haploid (25 %), four were diploid (50 %), and two were mixoploid (25 %) for haploid and diploid cells. Nine gynogenic plantlets showing abnormal development were diploid. Therefore, formation of gynogenic plantlets with abnormal phenotypes was likely due to genetic factors. Results obtained from this study suggest use of DH technology in the production of homozygous A. tuncelianum inbreeds in variety improvement programs.

Cryopreservation of Arecanut (Areca catechu L.) Embryogenic Calli by V Cryo-plate Method

Aims: Arecanut, a perennial palm species of Arecaceae family, has huge commercial value, and is grown mainly for its masticatory nuts. The ever-increasing demand for uniform quality plantlets from growers necessitates putting in place In vitro mass multiplication and other crop improvement programmes. The present study was carried out to standardize the procedure for cryopreservation of embryogenic calli of arecanut, derived from immature inflorescence cultures, by vitrification based cryo-plate technique. Study Design: Completely randomized design (CRD) with three replications. Place and Duration of Study: ICAR-Central Plantation Crops Research Institute, Kerala, India during 2019. Methodology: The embryogenic calli were precultured in Eeuwen's Y3 basal medium supplemented with sucrose (0.2, 0.3 and 0.4 M) for three days. Explants were affixed on cryo-plates and later dehydrated using plant vitrification solution 3 (PVS3) for 30 min. Cryoplates were inserted in cryovials and cryopreserved. Explants with no cryostorage served as control. Explants were rewarmed quickly in a water bath (40ºC) for 2 min and treated with unloading solution and cultured on recovery medium. Results: The results showed 8-10 % recovery of embryogenic calli that resulted in normal plantlet production. The clonal fidelity studies, using Start Codon Targeted (SCoT) marker, showed no variation of cryopreserved calli in comparison to the original calli. Conclusion: This preliminary study demonstrated the successful use of vitrification (V) cryo-plate technique in cryopreservation of embryogenic calli of arecanut. With better recovery percentage, the optimal concentration of sucrose in the preculture medium was found to be 0.3 M. Desiccation in PVS3 solution for 30 min had no adverse effect.

Improved in vitro rooting and acclimatization of “Violetta” Artichoke and “Green Globe” Artichoke

Artichoke (Cynara scolymus L.), a medicinal plant with high economic value, contains high levels of phenolic compounds; especially cynarine, which plays an important role in preventing cancer, cardiovascular disease, osteoporosis, diabetes and neurodegeneration, etc. Currently, Artichoke micropropagation has achieved some success; however, the rooting efficiency and plantlet quality are still limited. In this study, improving the quality of Artichoke plantlet related to the shoot quality and suitable substrates in in vitro rooting stage was studied on “Violetta” Artichoke (VA) and “Green Globe” Artichoke (GA). The results showed that shoots (1.5 cm) cultured on MS medium supplemented 0.5 mg/L KIN were most suitable to shoot multiplication of VA with the number of shoots/explant (3.67 shoots), number of shoots ≥ 2 cm (3 shoots); while, 1.0 mg/L BA was suitable to shoot multiplication of GA (5.33 shoots; 5.00 shoots; respectively) after 4 weeks of culture. Besides, the in vitro rooting was improved using 8 g/L commercial agar for VA; meanwwhile, 3 g/L gelrite for GA. In addition, the nylon bag culture system (120 mm × 250 mm) has potential in plantlet production (15 plants/bag) and can be applied for large scale micropropagation. In addition, VA and GA plantlets derived from in vitro culture gave the good acclimatization, growth and development after 8, 12 and 20 weeks cultivating at the green house conditions.

Long-Term Subculture Affects Rooting Competence via Changes in the Hormones and Protein Profiles in Cedrela Fissilis Vell. (Meliaceae) Shoots

Long-term subculture plays an essential role in the large-scale multiplication and production of somatic plantlets. We investigated the effects of long-term subculture on in vitro shoot development and ex vitro rooting associated with changes in the hormones and protein profiles in C. fissilis. The number of subcultures of shoots induced a decrease in the ex vitro rooting response. The reduction in adventitious root (AR) formation was associated with decreases in the contents of indole-3-acetic acid (IAA), abscisic acid (ABA), 12-oxo phytodienoic acid (OPDA), putrescine (Put), and spermine and increases in jasmonic acid (JA), jasmonoyl-isoleucine, trans-cinnamic acid, and salicylic acid contents in shoots at the fourth subculture compared to the first. The ornithine decarboxylase enzyme preferentially functions in the Put biosynthesis pathway and was related to the highest AR formation in shoots at the first subculture. Down-accumulation of the auxin-binding protein ABP19a in shoots from the fourth subculture compared to the first subculture was related to a decrease in both IAA contents and AR formation. In addition, down-accumulation of glucose-6-phosphate isomerase, glutamine synthetase leaf isozyme chloroplastic, 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, L-ascorbate peroxidase, cytosolic, monodehydroascorbate reductase, and 2-Cys peroxiredoxin BAS1-like, chloroplastic and up-accumulation of caffeoyl-CoA O-methyltransferase 1 and isoforms of peroxidase 4 proteins in shoots from the fourth relative to the first subculture were associated with a reduction in AR formation. These results showed that the understanding of hormonal and molecular mechanisms related to the potential of AR formation in shoots under successive subcultures is relevant to improving large-scale plantlet production in C. fissilis.

High-frequency direct somatic embryogenesis and plantlet regeneration from date palm immature inflorescences using picloram

Background Date palm (Phoenix dactylifera L.) is a traditional crop in arid and semi-arid areas. Its vegetative propagation can be achieved by offshoots, but possible number of offshoots in mother palm trees is limited. Micropropagation is a highly recommended strategy for obtaining date palm elite cultivars using shoot tip and immature inflorescences. In this study, micropropagation procedure using inflorescence explants of Medjool cv. is described. For culture initiation, explants from different spathe lengths were cultivated on Murashige and Skoog medium (MS) supplemented with picloram at 1.0 and 2.0 mg/l combined with 2iP at 0.5 mg/l alone and with both 2iP and BA at 0.25 mg/l for 24 weeks. The obtained direct globular embryos were transferred to maturation media with 0.1 mg/l picloram alone or combined with both 2iP and ABA separately and together for further development. Additionally, multiplication and rooting media were optimized by different cytokinins and auxins for high frequency of plantlet production. Acclimatization of in vitro plantlets was also investigated. Results The highest percentage of globular embryo formation was noticed with explants isolated from spathe lengths ranging from 10 to 15 cm. Addition of BA to initiation media with picloram encouraged a significant effect on embryonic culture formation percentage. Incorporation of ABA and 2iP to maturation medium was an effective factor for individual or multiple embryo emergence. Acclimatization of in vitro plantlets having 3–4 roots was successfully accomplished. Irrigation with the full strength solution (MS) encouraged the highest growth vigor degree, leaf number/plant, leaf width, root number, and root thickness degree of ex vitro plants. Conclusion This research provides an advanced regeneration system for large-scale production of date palm from immature inflorescences of Medjool cv. It opens up the prospects of using picloram with different growth regulators for rapid micropropagation of date palm.

The Effect of Zinc, Copper, and Silver Ions on Oat (Avena sativa L.) Androgenesis

Oat (Avena sativa L.) cultivars ‘Bingo’ and ‘Chwat’ were used to compare the embryogenesis competence of another culture. Despite the embryo-like structures obtained from both tested cultivars, only ‘Chwat’ produced green plantlets, which confirmed the cultivar dependency. ‘Chwat’ produced the highest number of embryo-like structures and green plantlets (0.7/100 anthers and 0.1/100 anthers, respectively). The embryo-like structure formation also depended on cold pretreatment combined with Cu2+, Zn2+, or Ag+ ion supplementation, which was applied during the tiller pretreatment or added to the induction media. The highest number of embryo-like structures (2.1/100 anthers) were observed on anthers derived from the tillers kept in a 50% Hoagland medium with the addition of 10 µM of CuSO4. In turn, the induction media supplemented with the ions Cu2+, Zn2+, or Ag+ increased neither the number of embryo-like structures nor the green plantlet production compared to the control conditions. However, such ion applications turned out to be most effective when the induction medium was enriched with 25 µM of AgNO3 and left to obtain the highest number of embryo-like structures and green plantlets (0.8/100 anthers and 0.2/100 anthers, respectively). Therefore, more attention should be paid to the possibilities of adjusting the media nutrient composition, as this may be the only way to significantly increase the efficiency of this method.

Aromatik Tıbbi Bitki olan Mentha x piperita L. ve Mentha pulegium L.’nin in vitro Kallus İndüksiyonu ve Mikroçoğaltım yoluyla Geliştirilmesi

In this study, we evaluate the potential for in vitro propagation of Mentha spp. for mass production. The two Mentha spp. (Mentha x piperita L., M. pulegium L.) were propagated with four successive 60-day subcultures in MS medium supplemented with for 100µL/L NAA (Naphthylacetic Acid) and 600µl/L IBA( Indole Butyric Acid). The shoots were rooted in the same media. The rooted plantlets were finally acclimatized in a growth room. Callus induction was carried out in MS (Murashige and Skoog) media supplemented with 100µL/L NAA and 250µL/L BAP (Benzylaminopurine). Callus was successfully induced from nodes of Mentha pulegium L. Through micropropagation, both Mentha spp increased in multiplication rates around 6-fold per month compared to the traditional propagation method. Mentha x piperita and Mentha pulegium showed the most significant potential for plantlet production through the micropropagation method.

How do clonal plantlets of mate respond to different substrate compositions and shading levels?

Mate (Ilex paraguariensis A. St.-Hil) is an arboreal species of great economic and socio-environmental importance in South American countries. This specie presents several difficulties during seminal propagation, and studies related to plantlet production by vegetative propagation are fundamental for obtaining homogenous mate plantations with high leaf productivity. Therefore, the present study aimed to evaluate the effects of substrate and shading levels on the morphophysiological quality of mate plantlets produced by mini-cuttings. Rooted mini-cuttings of four mate clones were cultivated on commercial substrate, subsurface soil and vermiculite (2:1:1 v/v/v), commercial substrate and subsurface soil (2:1 v/v) or subsurface soil, cattle manure and carbonized rice hulls (2:1:1 v/v/v). After 120 days, the plantlets were randomly distributed on benches to evaluate the effects of 50 and 80% shading screens. Regardless of the clone, the commercial substrate and subsurface soil composition allowed plantlet production with satisfactory development of both aerial part and root system at 120 days of cultivation. Clone 06SM17 produced plantlets with high averages of stem diameter, shoot height, number of leaves, total length, surface area, total volume of roots, and number of root tips. Both shade levels resulted in similar stem diameters, shoot heights, numbers of leaves, and a, b, and total chlorophyll indices. Clonal mate plantlets with satisfactory morphophysiological quality are produced in commercial substrate and subsurface soil (2:1 v/v) under 50 and 80% shading.

Young capitulum as important explant in in vitro mass propagation of gerbera (Gerbera jamesonii)

A new route of in vitro propagation of gerbera selected clones was successfully established using young capitula in tight buds and buds that were started to unfold stage as explant source. The one-fourth pieces of young capitula of tight flower stage and half-strength MS medium containing 0.25 mg/l BAP was the suitable for initiation and produced higher number of shoots per explant up to 3.8 shoots. The results were improved by culturing the one-fourth piece of 01.092 capitulums on MS medium fortified by 0.2 mg/l BAP and 0.02 mg/l NAA producing the highest shoot formation up to 8.5 shoots per explant with 28.7 leaves per explant and 2.1 cm leaf length. High multiple shoots were determined in third to fourth subculture periods and reduced thereafter with high multiplication rate noted on 01.092 clone. Shoots were easily rooted on half-strength MS medium supplemented with 2 g/l activated charcoal. Plantlets were transferred to ex vitro condition with 96.4% survivability of 03.045 clone using Cycas rumphii bulk and cocopeat (1:1, v/v) under spraying 1 g/l Growmore (32N:10P:10K) solution once week periodically. The route has high potential applied in qualified plantlet production for other Gerbera’s due to high shoots produced up to 35 shoots per whole young capitulum used.