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Doina Clapa, Alexandru Fira and Nirmal Joshee

This article presents a new acclimatization method, ex vitro acclimatization in float hydroculture. This protocol ensures the ex vitro acclimatization of the plantlets obtained in vitro in the rooting stage or the ex vitro rooting and acclimatization of the shoots obtained in the multiplication stage. Our hydroculture method is different from the techniques of flotation hydroculture because no fertilizers or plant growth regulators or other biostimulators are used and water oxygenation by bubbling is not provided. Ex vitro rooting and acclimatization in one stage without an in vitro rooting stage in floating cell trays was successfully carried out in Rubus fruticosus and Rosa hybrida cultivars, whereas ex vitro acclimatization of in vitro-rooted plantlets was successfully carried out in Rubus idaeus, Ribes nigrum, Prunus cerasus × P. canescens, Lycium barbarum (Goji berry), Amelanchier canadensis, Drosera rotundifolia, Drosera capillaris, and Nephrolepis sp. In another set of experiments, the floating cell trays were replaced with layers of floating perlite and the latter method was also tested for non-rooted shoots obtained in vitro in the multiplication stage or rooted plantlets obtained in the rooting stage. Direct ex vitro rooting in floating perlite was successful in ‘Tayberry’ (Rubus fruticosus × Rubus idaeus) (78.12% rooting); Amelanchier canadensis, Rubus fruticosus ‘Chester’, Rubus idaeus ‘Erntesegen’, Vaccinium macrocarpon, and Vaccinium corymbosum with rooting percentages above 80%; and Rubus idaeus ‘Willamette’ and Rosa hybrida ‘Cristiana’ with rooting percentages above 80%.

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Nirmal Joshee*, Bipul K. Biswas and Anand K. Yadav

Centella asiatica L. (Apiaceae family), also called `Indian Pennywort,' is a prostrate, faintly aromatic, and stoloniferous perennial herb with long petiolated leaves. In the Ayurvedic medicine, it is reputed as a nervine tonic along with antibacterial, antifeedant, antileprotic and wound healing properties. Centella contains glycosides, indocentelloside, brahmoside, and asiaticoside. Its leaves are rich in carotenoids and vitamins B and C. In vitro culture techniques which offer a viable tool for mass propagation of plants have recently become increasingly popular for conservation of rare, endangered and threatened medicinal plants germplasm. Centella tissue culture has been reported to experience high incidences of microbial contamination which drastically reduces survival of explants. Thus, the main purpose of this study was to develop an efficient micropropagation technique for Centella asiatica to reduce explant contamination and rapidly disseminate superior clones for research and production. Here we present induction and further development of somatic embryos, using Centella stolons as explants. Somatic embryos were induced in response to 2,4-D shock on MS medium. Initially, somatic embryos appeared as highly nodular callus and eventually developed into somatic embryos that exhibited globular, heart shaped and cotyledonary stages. After auxin shock, cultures were regularly transferred to MS basal medium where somatic embryos completed various developmental stages and then germinated to give rise to new plantlets. In this presentation, we will demonstrate complete protocols for the successful sterilization of Centella explants prepared from plants that had abundance of fungal and bacterial contamination.

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Bipul K. Biswas*, Nirmal Joshee and Anand K. Yadav

Guava (Psidium guajava L.), also called `apple of tropics,' is immensely nutraceutical and horticulturally important. Being a tropical plant, it cannot stand temperatures below 25° F and needs frost protection to grow in temperate regions. To adapt in cold climate, cold hardy guava cultivars are needed. Conventional ways are uneconomic in time and efforts. Still, transgenic plants developed using biotechnological approaches of tissue culture and rDNA technology, appear to have great potential. Thus, protocols for in vitro propagation of guava were developed via organogenesis and somatic embryogenesis using nodal explants from mature trees and young zygotic embryos, respectively. Nodal explants induced multiple shoots when cultured on MS medium fortified with KIN, BAP and Ad.S. Adding a (NO3)2 to medium was useful to prevent in vitro shoot tip browning of adventitious shoots. Rocker liquid culture greatly increased growth of multiple shoots compared to the agar-based medium. It appears to be a good tool for woody plant tissue culture. Induction of somatic embryos in guava was also achieved on MS medium supplemented with IAA auxin. About 80% to 90% somatic embryos germinated normally. To achieve Agro-bacterium-mediated gene transfer in guava, on-going co-cultivation of organogenic tissues of guava is to optimize protocols for freeze tolerance gene (CBF1, CBF2, CBF3) transfer. Plasmid vectors containing selectable markers (nptII gene for antibiotic selection and GUS reporter gene as scorable gene mediated selection), with CaMV 35S promoter gene has been introduced into guava tissues and the resultant plants showed antibiotic resistance. Details of the experimental procedures and up-to-date results will be discussed.

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Nirmal Joshee, Bipul K. Biswas and Anand K. Yadav

Past research experience with Centella asiatica micropropagation suggests a very high rate of contamination during the culture establishment stage. We demonstrate protocols for successful sterilization of Centella explants prepared from field-grown plants with an abundance of fungal and bacterial contamination. Sequential steps during sterilization and explant preparation process included a dip for 30 s in 70% ethyl alcohol, weak bleach treatment for 12 min, and a 60-min soak in plant preservative mixture before establishing cultures. We also report a reproducible system for somatic embryogenesis in Centella using leaf and stolon tip explants collected from naturally growing populations. Somatic embryos were induced within 3 to 4 weeks of culture in the dark on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). Initial embryogenic mass appeared as nodular callus, which eventually developed into actual somatic embryos exhibiting globular, heart-shaped, and cotyledonary stages. Leaves produced embryogenic calli at 2.26 and 4.52 μm 2,4-D, whereas stolon tips were responsive only in the 9.04 μm 2,4-D treatment. Withdrawal of 2,4-D/growth regulators from the induction medium resulted in the maturation and further development of the embryos into plantlets. Regular subculturing of the embryogenic calli into MS medium sustained their regenarability for more than 1 year. Somatic embryos were individually encapsulated in sodium alginate and calcium chloride-based encapsulation matrix to produce artificial or synthetic seeds (synseeds). Synseeds with 2% sodium alginate were found best for the survival and germination recorded after their storage at 5 to 8 °C for 30 and 60 days. We report protocols for C. asiatica to reduce explant contamination before establishment of cultures on somatic embryo induction medium and efficient somatic embryogenesis to facilitate conservation and mass production of elite germplasm. This may further assist rapid dissemination of superior clones needed for research and commercial production.

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Wei Hao, Rajeev Arora, Anand K. Yadav and Nirmal Joshee

Guava (Psidium guajava L.) is a tropical evergreen tree that tolerates a wide range of frost-free environments. In recent years, the American market demand for exotic and nutritious fruits, like guava, has been increasing, and, with a long harvest period, guava can be a potential alternative, high-value cash crop in the United States. However, the major limitation with commercializing guava cultivation in the United States is its low cold tolerance. In this article, we studied the physiology of freezing tolerance and cold acclimation in guava. Laboratory freeze–thaw tests (on leaves), shoot growth and leaf relative water content measurements, leaf anthocyanin content analyses, and leaf protein analyses were performed on nonacclimated and cold-acclimated guava cultivars Lucknow-49 and Ruby × Supreme. The leaf freezing tolerance (expressed as LT50 values) of nonacclimated tissues was ≈–2.5 °C and significantly enhanced to ≈–4.4 °C after an environmentally controlled cold acclimation regime for both cultivars. However, when compared based on actual injury sustained by leaves at various freezing temperatures in a freeze–thaw test, ‘Ruby × Supreme’ exhibited significantly less injury than ‘Lucknow-49’ at most temperatures. Growth and leaf relative water content reduced, whereas leaf anthocyanins accumulated during cold acclimation. Leaf protein analyses, which were performed after cold acclimation and drought stress, revealed that four proteins (69, 48, 23.5, and 17.4 kDa) accumulated in response to low temperatures, and two proteins (17.4 and 16 kDa) accumulated in response to drought stress. Antidehydrin immunoblots revealed that one common 17.4 kDa dehydrin accumulated in response to cold and drought stresses. Our data indicate that guava possesses leaf freezing tolerance, exhibits cold acclimation ability, and that ‘Ruby × Supreme’ leaves are relatively more freezing-tolerant than ‘Lucknow-49’ when compared up to –4 and –8 °C for nonacclimated and cold-acclimated tissues, respectively. Cold acclimation in guava appears to be a multifactorial process involving complex physiological and biochemical changes and also overlapping responses with drought stress.

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Bipul Biswas, Nirmal Joshee, Ashish Yadav and Anand K. Yadav

Phalsa[Grewia asiatica (L.) Tiliaceae] is an exotic fruit with good nutraceutical values. It cannot be grown in temperate climates with severe winters. Therefore, genetic improvement of phalsa for cold tolerance is essential. In order to apply biotechnology through genetic transformation to enhance cold hardiness, a reliable and rapid micropropagation system is needed. Thus, developing the most dependable micropropagation protocols for phalsa was the primary goal of this research. Phalsa explants prepared from different tissues, including leaf, nodes, internode, and zygotic embryos, were collected from mature trees growing in the specialty plants house, cultured on MS medium supplemented with various cytokinins alone or along with auxins and incubated under a 10-hour photoperiod at ambient temperature. In vitro propagation of phalsa tissues through both organogenesis and somatic embryogenesis was achieved. Of these, single shoots were developed from nodal explants as a result of budbreak on MS medium supplemented with BAP, kinetin, and zeatin separately. Somatic embryos were developed from the zygotic embryos when cultured on MS medium with 0.023 μm BA + 0.022 μm zeatin, for 2 weeks following a pulse treatment on NN medium supplemented with 5% sucrose, 0.11 μm BAP, 0.22 μm 2,4-D, and 29.20 μm L-glutamine. Somatic embryogenesis was also observed on modified basal medium supplemented with 13% sucrose, 58.40 μm L-glutamine, and 1.75 μm IAA. Enormous callusing was a major problem for in vitro studies with this species, irrespective of media composition. Further studies for multiple shoot development and higher frequency of SE induction are under way.

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Arsène Similien, Dennis A. Shannon, C. Wesley Wood, Edzard van Santen, Nirmal Joshee and Wheeler G. Foshee

American skullcap (Scutellaria lateriflora L.), a medicinal plant species valued for its sedative properties associated with flavonoids, is generally harvested from the wild. Scientific information on how field cultivation practices affect dry matter yield is lacking in this species. A 2 × 2 × 3 split plot factorial experiment within a randomized complete block design was conducted on a Marvyn loamy sand (fine-loamy, kaolinitic, Thermic Typic Kanhapludults) in Central Alabama to explore effects of light, irrigation, and nutrient application on dry matter yield of American skullcap. Treatment factors were shade (40% shade vs. no shade), irrigation (applied at 30 kPa vs. no irrigation), and nutrients [no added nutrients vs. nutrients added as chemical fertilizer (100 kg nitrogen, 68 kg phosphorus, 42 kg potassium/ha) or chicken litter (100 kg nitrogen, 50 kg phosphorus, and 123 kg potassium/ha)]. Shade formed the main plot units, whereas irrigation × nutrient factorial combinations were subplots. Skullcap shoots in experimental plots were harvested four times during the course of the two-year experiment (2007, 2008). All growth variables measured, except percent dry matter, performed better under shade than in full sun. Dry matter yield was increased 45% by shade, 61% by irrigation, and 22% by addition of nutrients. A significant irrigation × nutrients interaction was observed at the first and second harvests. Highest yields were obtained with the irrigation + manure and irrigation + fertilizer treatments under shade and the lowest with fertilizer and the control treatments in full sun.

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Ayse Tascan, Jeff Adelberg, Mevlut Tascan, Agnes Rimando, Nirmal Joshee and Anand K. Yadav

Three Scutellaria species (Scutellaria lateriflora, S. costaricana, and S. baicalensis) were grown in different in vitro physical environments: agar, liquid culture, and liquid culture with fiber-supported paper (with initial media volumes of 20 mL and 30 mL). During an 8-week time course, tissue growth was assessed for each species by fresh weight (FW), dry weight (DW), percent DW, and multiplication ratio. Water use and hyperhydricity were also compared. Scutellaria lateriflora plantlets grown in liquid were hyperhydric despite the greatest accumulation of dry mass, but multiplication diminished with time as plants became hyperhydric. In contrast, S. costaricana and S. baicalensis plantlets had higher FW and DW on agar. With all Scutellaria species tested, plantlets grown on agar or fiber-supported paper were not hyperhydric, and fiber-supported paper with 20 mL initial volume yielded plants with the greatest percent DW. The lowered hyperhydricity was related to reduced water uptake. The flavonoids baicalin, baicalein, and wogonin were quantified in plants grown on fiber-supported paper culture. The baicalin concentrations in in vitro cultured S. lateriflora shoots was comparable to those of field-grown plants. The in vitro method presented a unique opportunity to enhance baicalein content and produce wogonin-rich roots. S. costaricana plantlets in vitro showed high levels of the three flavonoids compared with S. baicalensis and S. lateriflora. Growing non-hyperhydric tissues on fiber-supported paper, in vitro, allowed the clonal propagation of Scutellaria species with increased flavonoid content to proceed in a simple, controlled environment.