In vitro Regeneration of Japanese Burdock (Arctium lappa L.) from Cotyledon and Leaf Derived Explants

Considering the vegetable and medicinal values, a micropropagation protocol has been established for Japanese Burdock (Arctium lappa L.) by culturing the explants of cotyledon and leaf obtained from in vitro grown seedlings. Direct shoot regeneration was achieved from cotyledon and leaf explants on MS fortified with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. In addition, both the explants also formed callus from their cut margins within 6 weeks of cultivation on medium complemented with 6.0 μM BAP and 4.0 μM IBA or NAA. Adventitious shoots were also redeveloped through indirect organogenesis from the cotyledon and leaf-derived callus within 10 weeks of culture on MS containing 4.0 μM BAP and 2.0 μM IBA or NAA. The highest rate of shoot reproduction was attained at the third subculture, and more than 12.6 shoots were formed per callus clump. Within 4 weeks of transfer to the rooting medium on MS containing 6.0 μM IBA, the cultured micro-shoots produced highest 5.3 roots per cultured shoot. Rooted plantlets were successfully established on a soil-composed-sand mixture under natural condition with 93.3% survival rate Plant Tissue Cult. & Biotech. 31(2): 123-134, 2021 (December)

In vitro mutagenesis in anthurium induced by colchicine

Developing new varieties of anthurium by hybridization can take 8-10 years; therefore, induced mutagenesis can be an alternative strategy to hybridization. The objective of this work was to induce mutations in A. andreanum by exposing explants obtained from vitroplants to colchicine. Explants of leaves, nodes and roots obtained from vitroplants were exposed to 0.1 % colchicine for 0, 2, 3 and 4 h. The mean lethal dose (LD50), survival, number of explants that generated callus, number of explants that formed shoots and the number of shoots per explant were evaluated. The karyotype of the presumed mutated regenerated plants was determined by the root apex squash technique. The leaves showed the highest sensitivity to cochicine. The survival of the root explants treated with colchicine was 100 %; 4 % of roots exposed for 2 and 3 h formed adventitious shoots (120 shoots). For nodes, the LD50 was found at 3.98 h; 76 and 56 % of the nodes cultivated for 2 and 3 h with colchicine formed adventitious shoots (4.4 and 3.6 shoots). The plants regenerated from the explants exposed to colchicine showed morphological changes. The chromosomal number of the regenerated vitroplants from the explants exposed for 2 and 3 h to colchicine was 2n = 29, while that of those obtained from the explants that remained on the colchicine for 4 h was 2n = 31. The sensitivity to colchicine was a function of the type of explant and the dose used. Colchicine caused the loss (monosomy) or gain of chromosomes (trisomy).

Use of Biotechnological Methods to Support the Production of New Peach Hybrids

Peach [Prunus persica (L.) Batsch] is among the most demanded fruit crops in the world. Biotechnological methods help to originate new hybrid forms in order to increase the cultivar diversity and create new valuable genotypes. Cross combinations between the cultivars Clyde Wilson, Jerseyglo, Loadel, Summerglo and the promising cultivar ‘Nikitskiy Podarok’ have been done. The embryos of these hybrids germinated and formed plantlets after stratification at 4 °C for 45–60 days. The best regeneration rates in the hybrids ‘Loadel’ × ‘Nikitskiy Podarok’ and ‘Summerglo’ × ‘Nikitskiy Podarok’ (96.30% and 92.59%, respectively) were noted on hormone-free Monnier culture medium supplemented with 400.0 mg L−1 casein hydrolyzate. When the newly formed plantlets had necrosis of the shoot apex or immature roots, nodal shoot segments were used. At the same time, a high regeneration capacity was noted in the hybrids ‘Summerglo’ × ‘Nikitskiy Podarok’ and ‘Loadel’ × ‘Nikitskiy Podarok’ on B5 culture medium with 0.75 mg L−1 6–benzyl–aminopurine (BAP) + 0.1 mg L−1 indole–3–butyric acid (IBA). After the second subculture, the number of new adventitious shoots was 5.18 ± 0.18 and 4.95 ± 0.18 shoots per explant, respectively. The plants obtained from the hybrid embryos in a soil mixture soil: peat: sand (3:1:1) were adapted. The main morphological and anatomical features of the leaf blades in newly originated peach hybrids have been studied: the thickness of their tissues and the distribution of stomatal apparatus, as well as the physiological parameters of the photosystem II activity in regenerants cultured in vitro and during their in vivo acclimatization. The high capacity to post aseptic adaptation in the obtained hybrids has been shown.

High frequency plant regeneration of Chrysopogon zizanioides via organogenesis and somatic embryogenesis-an underutilized pharmaceutically valuable biofuel plant

Vetiver (Chrysopogon zizanioides) is an essential oil-producing plant that has tremendous application in cosmetics, perfumery, and herbal medicine. Natural sterility and indiscriminate harvests lead to the risk of extinction of plant species in natural habitats. Therefore, a protocol for regeneration systems via organogenesis and somatic embryogenesis using node, leaf, and root explants has been standardized. The highest shoot regeneration frequency (72.2%) through organogenesis was attained from node explants on MS (Murashige & Skoog) medium comprising 2.0 mg L-1 BAP (“6-benzylaminopurine”). Concurrently, leaf explants cultivated on MS medium augmented by 1.0 mg L-1, 2, 4-D (“2, 4-dichlorophenoxyacetic acid”) formed the optimal frequency (75.35%) of white friable compact (WFC) callus. However, the root explant was less responsive for WFC callus induction. Organogenic WFC callus cultivated on MS medium fortified by kinetin (1.0 mg L-1) as well as BAP (1.0 mg L-1) revealed the highest shoot regeneration efficiency (75.49%) with 48 shoots per callus. Adventitious shoots obtained from node and WFC callus of both leaf and root explants cultivated on MS medium increased by NAA (2.0 mg L-1 showed the optimal rooting of 76.97%. Concomitantly, an elevated frequency of somatic embryogenesis (52.50%) was recorded from leaf explants on MS medium using BAP (0.5 mg L-1) & 2, 4-D (1.0 mg L-1). Leaf explants were superior to node and root explants for somatic embryo initiation. The cotyledonary embryos were efficiently germinated into complete plantlets on a hormone-free MS medium. The plantlets gathered from organogenesis & somatic embryo genesis was effectively acclimatized into phenomenally similar plants. This technique may be applicable for wide-range propagation, genetic engineering, and the formation of bioactive compounds.

Micropropagation of Rare Scutellaria havanensis Jacq. and Preliminary Studies on Antioxidant Capacity and Anti-Cancer Potential

We report the development of in vitro propagation protocols through an adventitious shoot induction pathway for a rare and medicinal Scutellaria havanensis. In vitro propagation studies using nodal explants showed MS medium supplemented with 10 µM 6-Benzylaminopurine induced the highest number of adventitious shoots in a time-dependent manner. A ten-day incubation was optimum for shoot bud induction as longer exposures resulted in hyperhydricity of the explants and shoots induced. We also report preliminary evidence of Agrobacterium tumefaciens EHA105-mediated gene transfer transiently expressing the green fluorescent protein in this species. Transformation studies exhibited amenability of various explant tissues, internode being the most receptive. As the plant has medicinal value, research was carried out to evaluate its potential antioxidant capacity and the efficacy of methanolic leaf extracts in curbing the viability of human colorectal cancer cell line HCT116. Comparative total polyphenol and flavonoid content measurement of fresh and air-dried leaf extract revealed that the fresh leaf extracts contain higher total polyphenol and flavonoid content. The HCT 116 cell viability was assessed by colorimetric assay using a 3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide, showed a steady growth inhibition after 24 h of incubation. Scanning electron microscopy of leaf surface revealed a high density of glandular and non-glandular trichomes. This research provides a basis for the conservation of this rare plant and future phytochemical screening and clinical research.

Micropropagation of Rare Scutellaria Havanensis Jacq. And Preliminary Studies on Antioxidant Capacity and Anti-cancer Potential

We report the development of in vitro propagation protocols through adventitious shoot induction pathway for a rare and medicinal Scutellaria havanensis. In vitro propagation studies using nodal explants showed MS medium supplemented with 10µM 6-Benzylaminopurine induced highest number of adventitious shoots in a time dependent manner. A ten - day incubation was optimum for shoot bud induction as longer exposures resulted in hyperhydricity of the explants and shoots induced. We also report preliminary evidence of Agrobacterium tumefaciens EHA105 - mediated gene transfer transiently expressing of green fluorescent protein in this species. Transformation studies exhibited amenability of various explant tissues, internode being the most receptive. As the plant has medicinal value, research was carried out to evaluate its potential antioxidant capacity and the efficacy of methanolic leaf extracts in curbing the viability of human colorectal cancer cell line HCT116. Comparative total polyphenol, and flavonoid content measurement of fresh and air dried leaf extract revealed that the fresh leaf extracts contain higher total polyphenol and flavonoid content. The HCT 116 cell viability assessed by colorimetric assay using a 3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide, showed a steady growth inhibition after 24 hours of incubation. Scanning Electron Microscopy of leaf surface revealed high density of glandular and non-glandular trichomes. This research provides basis for the conservation of this rare plant and future phytochemical screening and clinical research.

Mass Propagation of Nauclea diderrichii (de Willd. & Th. Dur) Merr. Seedlings through Tissue Culture Technique

Nauclea diderrichii is a tree species of economic importance. However, its plantation establishment is limited by inadequate seedling production. Hence, there is ample scope of tissue culture for its mass propagation. Its in vitro plantlets development as affected by media strengths indicated that 100 % seed germination was obtained in full MS basal medium while the least (3.35 %) was from quarter-strength at 8 Weeks after inoculation (WAI). The effects of BAP and NAA assessed on the growth of its sub-cultured plantlets showed that highest number of leaves (17) and adventitious shoots (3) were obtained from MS basal medium supplemented with 0.1 mg/l BAP only. Whereas, highest shoot length (3.61 cm) and average number of roots (5/plantlet) were obtained from the same medium without hormone(s) at 8 WAI. Further sub-culturing into MS with 0.05 mg/l NAA resulted into plantlets having optimum shoot and massive root growth ready for acclimatization in 6 WAI. The plantlets were successfully acclimatized using coconuthusk/ topsoil mixture with 90 % survival. Plant Tissue Cult. & Biotech. 31(1): 51-60, 2021 (June)

Protocols for Adventitious Regeneration of Amelanchier alnifolia var. cusickii and Lonicera kamtschatica ‘Jugana’

The aim of this work was to assess the regeneration capacity of Amelanchier alnifolia var. cusickii and Lonicera kamtschatica cv. ‘Jugana’ from different types of explants under various hormonal treatments. The whole leaves, petioles, and internodal segments of in vitro plants were examined as explants. Several plant growth regulators (cytokinins and auxins) were evaluated for their ability to induce adventitious regeneration. Direct and indirect organogenesis was achieved under certain culture conditions in both species. The frequency of shoot regeneration was strongly dependent on concentrations of plant growth regulators in the induction media (L.kamtschatica ‘Jugana’) or concentrations of plant growth regulators in the induction media and type of explant (A. alnifolia var. cusickii). Results showed that leaves were not suitable explants for A. alnifolia var. cusickii. Both species were able to regenerate shoots from internodal segments and petioles. The highest induction of shoots was obtained on Murashige and Skoog (MS) medium enriched with 2 mg/L thidiazuron (TDZ) and 0.5 mg/L indole-3-butyric acid (IBA) for Amelanchier alnifolia and with 1 mg/L TDZ and 0.2 mg/L indole-3-acetic acid (IAA) for L. kamtschatica ‘Jugana’. Obtained adventitious shoots were further proliferated in order to investigate their multiplication capacity. The multiplication of shoots was successful in all cultivars, with the best results reported in A. alnifolia var. cusickii (7.07 shoots/explant on average).

High-Efficiency Organogenesis and Evaluation of the Regenerated Plants by Flow Cytometry of a Broad Range of Saccharum Spp. Hybrids (Sugarcane)

The aim of this study was to evaluate the organogenic potential of Brazilian sugarcane varieties, in addition to verifying the in vitro multiplication rate and genetic stability by flow cytometry over monthly and consecutive subcultures. Thus, stem apices of twenty-two varieties were collected in the field and taken to the laboratory where external layers of leaves were removed. After disinfection, the achlorophyllous portion was sectioned and placed on MS medium with 5.0 mg L-1 NAA and 0.5 mg L-1 KIN. For multiplication rate assessments, ten varieties were selected and inoculated in of liquid or semi-solid MS medium with 0.10 mg L-1 KIN and 0.20 mg L-1 BAP, and subcultured every 30 days for a period up to 8 months. Genetic stability was verified by flow cytometry every two subcultures. At the end of the experiment, sprouts were rooted and acclimatized in a greenhouse. Regeneration occurred both by direct and indirect organogenesis, and the sugarcane varieties differed significantly in their regeneration capacity and number of adventitious shoots formed. For multiplication, a significant interaction was observed between variety, consistency of the culture medium and number of subcultures. In general, in the first subcultures, the liquid medium gave similar or better results compared with semi-solid medium; however, from the fourth transplanting onward, the semi-solid medium was superior. Morphological variations were verified from the fourth subculture. In addition, in some varieties, small changes in the relative DNA content were detected by flow cytometry. Sprouts of normal-looking sugarcane were successfully rooted and acclimatized after the eighth subculture.

In Vitro Root Induction from Argan (Argania spinosa (L.) Skeels) Adventitious Shoots: Influence of Ammonium Nitrate, Auxins, Silver Nitrate and Putrescine, and Evaluation of Plantlet Acclimatization

Argania spinosa (L.) Skeels is an endangered plant species endemic to Morocco. In recent years, attempts to develop in vitro regeneration systems for this species were made. However, rooting and acclimatization of in vitro plants have been a bottleneck for successful propagation. In the present study, the effects of different concentrations of auxins, putrescine, silver nitrate (AgNO3) and ammonium nitrate on the in vitro rooting of adventitious shoots of two argan genotypes “Mejji” and “R’zwa”, were evaluated. The highest rooting percentages (86.6% in “Mejji” and 84.4% in “R’zwa”) were observed on Murashige and Skoog (MS) medium modified by reducing the ammonium nitrate concentration and supplemented with 1.5 mg L−1 indole-3-butyric acid (IBA), 0.5 mg L−1 1-naphthalene acetic acid (NAA), 2 mg L−1 AgNO3 and 160 mg L−1 putrescine. This medium resulted in the development of a good root system after only 10 days of culture. Plantlet acclimatization was carried out using different substrate mixtures, and high survival rates (100%) were observed when the substrate contained either peat alone or a sand–peat mixture (1:1, w/w). The high percentages of rooting and acclimatization reported in the present study are of high importance for rapid and large-scale propagation of this endangered species.