In Vitro Cellular & Developmental Biology

In vitro cloning via direct somatic embryogenesis and genetic stability assessment of Paphiopedilum niveum (Rchb.f.) Stein: the endangered Venus's slipper orchid Abstract Wild populations of Paphiopedilum niveum (Rchb.f.) Stein, an endangered orchid species, have been threatened with extinction due to climate change and overcollection. Conventional methods of propagation are unable to meet market demands. The current study reports the production of genetically stable, protocorm-derived protocorm-like bodies (PLBs) of P. niveum that can be used for commercial production. These PLBs are referred to hereafter as somatic embryos (SEs). Factors affecting SE induction and SE proliferation were examined. The highest percentage of SE formation (68.33 ± 11.77%) and the maximum number of SEs per explant (5.19 ± 0.67) were obtained on modified Vacin and Went (MVW) medium containing 0.1 mg L−1 1-naphthaleneacetic acid (NAA). The highest increase in fresh weight (FW) of proliferated SEs (183.33 ± 28.93 mg per 100 mg initial FW) was gained on hormone-free MVW. These SEs eventually developed into vigorous plantlets after culturing on MVW supplemented with 0.2% (w/v) activated charcoal and 50 g L−1 banana homogenate for 12 wk. A histological study revealed that SEs originated from both the epidermal and the sub-epidermal layers of the original protocorm via direct somatic embryogenesis. The genetic homogeneity between the mother generation (V1; the original protocorm) and subsequent generations (V2 and V3; the primary and secondary somatic embryos, respectively) was shown to be identical via random amplification of polymorphic DNA (RAPD) assessment. All regenerated plants grew well under greenhouse conditions.

Micropropagation of Nelumbo nucifera 'Weishan Hong' through germfree mature embryos Abstract Asian lotus (Nelumbo nucifera Gaertn.) is an economically important aquatic plant that is widely cultivated in Asia. It is mainly propagated by dividing rhizomes, which maintains genotype stability but is also laborious and time-consuming. An efficient in vitro method that ensures genetic stability is highly desirable. An optimized method was developed to produce intact and sterile mature embryos by soaking fruits in sterile water containing 2% (v/v) plant preservative mixture. Using mature embryos as explants, an improved micropropagation procedure was established in N. nucifera 'Weishan Hong.' The growth patterns of explants differed with the types and concentrations of cytokinins. Media containing 0.5 to 2.0 mg L−1 kinetin were suitable for elongation of unexpanded stems, while media with 1.0 to 2.0 mg L−1 6-benzylaminopurine (BAP) the stems were shorter. Higher concentrations of about 3.0 mg L−1 BAP promoted shoot cluster formation. Liquid–solid, double-layer media promoted plantlet growth. The well-rooted plantlets could normally grow after being transplanted. Lotus is known to be recalcitrant to regeneration from callus. Apices of mature embryos, aseptic plantlets, and young embryos formed callus successfully when the medium was supplemented with 2,4-dichlorophenoxyacetic acid and thidiazuron. Callus morphology differed with respect to the source. However, only a few of the calluses developed axillary shoots.

Increased freezing stress tolerance of Nicotiana tabacum L. cv. Bright Yellow-2 cell cultures with the medium addition of Ascophyllum nodosum (L.) Le Jolis extract Abstract The study of bioactive components of the brown alga Ascophyllum nodosum (L.) Le Jolis has shown that they promote growth and increase productivity of plants. However, a standardized model system providing consistent responses to such bioactive components has yet to be established. Given that freezing stress, especially in northern climates, reduces plant growth and productivity, a technique was developed to protect plant cells under freezing stress using the natural products made from the abundant A. nodosum as a cell culture supplement. In this study, a homogenous cell culture system of Nicotiana tabacum L. cultivar Bright Yellow-2 (BY-2) suspension cells was used to investigate the bioactivity and protection level of this alga extract under freezing temperatures, and BY-2 cell growth, physiology, and molecular changes were measured in the presence or absence of the extract. The results indicated that A. nodosum extract significantly improved BY-2 cell survival after exposure to freezing temperatures. Inclusion of alga extract also improved cell growth, membrane stability, and nuclear integrity, and reduced cell death of cold-stressed BY-2 suspension cultures. It was concluded that A. nodosum extract influenced cellular and molecular regulation and triggered mechanisms, such as osmolyte accumulation and antioxidant activity, to combat freezing stress in BY-2 suspension cells.

Goji berry ( Lycium barbarum L.) in vitro multiplication improved by light-emitting diodes (LEDs) and 6-benzylaminopurine Abstract In Brazil, Lycium barbarum L. (goji berry) is an agronomically valuable, imported, commercialized plant. The present study evaluated the effects of cytokinins and light sources on optimization of in vitro multiplication of goji berry by axillary bud proliferation. Nodal explants were excised from plants 90 d after in vitro germination and cultured on Murashige and Skoog (MS) medium containing 30 g L−1 sucrose; 7 g L−1 agar-agar; and 0, 2, 4, 8, or 16 μM 6-benzylaminopurine (BAP), 6-furfurylaminopurine (KIN), or thidiazuron (TDZ). After 60 days, regenerated lateral buds and shoot lengths were evaluated. The regenerated shoots were inoculated in MS supplemented with 0.0, 2.5, 5.0, 10.0, or 20.0 μM BAP under white fluorescent lamps or mixed treatment of red/blue (RB) light-emitting diode (LED) lamps. After 60 days in vitro cultivation, the number of formed shoots, leaf number, and shoot length were determined. Subsequently, the shoots were separated and acclimatized in a greenhouse for 30 d and the percentage of survival was evaluated. High regeneration and shoot numbers during lateral bud regeneration occurred with MS medium plus 4 μM BAP. The estimated optimum concentration of 11.56 μM BAP under RB LED lamp resulted in a high shoot number (8.15), with more average leaves per plant (9.2) at 5 μM BAP, and longer shoots (12.34 cm) at 12.42 μM BAP. Acclimatization was successful with 100% survival of ex vitro plants. An efficient in vitro multiplication protocol of an economically important species, goji berry, was developed to facilitate large-scale commercialization of this species.

Wounding and medium formulation affect de novo shoot organogenic responses in hypocotyl-derived explants of annatto ( Bixa orellana L.) Abstract Bixa orellana L. (annatto) is the unique exploited source for the production of bixin and norbixin natural dyes, which are widely used in the food, pharmaceutical, cosmetic, and textile industries. In tissue culture, adequate culture conditions are essential for the development of the plant. Among them, the saline composition of the culture medium and the type of explants are limiting factors. In this study, the effect of MS (Murashige and Skoog), Correia and colleagues (JADS) medium, Woody Plant Medium (WPM), and Driver Kuniyuki Walnut (DKW) culture media and wounding of hypocotyl explants on de novo shoot organogenesis (DNSO) of annatto were evaluated. It was demonstrated that JADS medium improved the nutritional status of shoots, which promoted the best development and quality of the plants generated after acclimatization, when compared to MS medium. The use of JADS medium for annatto DNSO during 45 d of culture was sufficient to regenerate and stimulate the growth of shoots, which had adequate elongation and rhizogenic responses. In addition, enlargement of the wound surface, using longitudinal sections of the hypocotyl explant cut surface down on JADS medium, significantly improved the frequency and number of newly formed shoots. This protocol enabled production of an average of 17 30-mm-long shoots per explant after 90 d of culture. These results will be useful for future studies related to annatto biotechnology.

Evaluation of canavanine as an allelochemical in etiolated seedlings of Vicia villosa Roth: protoplast co-culture method with digital image analysis Abstract Strong allelopathic activities of etiolated seedlings of a leguminous plant, Vicia villosa Roth (hairy vetch), were confirmed by a protoplast co-culture method. The combination of 1% Cellulase RS and 1% Driselase 20 in 0.4 M mannitol solution was used for protoplast isolation. The inhibitory effects of V. villosa protoplasts on the growth of recipient Lactuca sativa L. (lettuce) protoplasts were determined using 96-well culture plates with 50 μL of liquid medium consisting of Murashige and Skoog's basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM 6-benzyladenine, 3% (w/v) sucrose, and 0.4 M mannitol in each well. The allelopathic activity of V. villosa epicotyl protoplasts (2 × 102 to 105 mL−1), on cell division of lettuce protoplasts (6 × 103 to 5 × 104 mL−1), was stronger than that of roots and was stronger than on cell wall formation. Digital image analysis of co-cultured protoplasts (DIA-PP method) showed the effect of V. villosa protoplasts on the accumulation of a yellow color in lettuce protoplasts. Weaker inhibition was seen than at the cell wall formation and cell division stages. Effects of putative allelochemical, canavanine at 10 μM strongly inhibited division of lettuce protoplasts, whereas putative allelochemical, cyanamide was stimulatory at 10 μM, but inhibitory at 1 mM. A high level of canavanine was found in epicotyl protoplasts using gas chromatography-mass spectrometry. Canavanine was suggested to be the allelochemical in V. villosa epicotyl protoplasts of etiolated seedlings, although its effect differed with the growth stages of lettuce protoplasts.

A new temporary immersion system for commercial micropropagation of banana ( Musa AAA cv. Grand Naine) Abstract Temporary immersion systems (TIS) are among the best options for commercial micropropagation. However, although successes have been reported with TIS, it is necessary to establish the most suitable system. The aim of this research was to compare the efficiency of different banana (Musa AAA cv. Grand Naine) micropropagation systems. In addition, a liquid medium with partial immersion and a semi-solid culture medium were evaluated. At 28 d of culture, the number and length of shoots, number of leaves per shoot, chlorophyll content, stomatal index, and the percentage of closed stomata were recorded. Additionally, the survival percentage and ex vitro development of the plantlets were evaluated during acclimatization. Results showed that TIS have a higher multiplication rate with respect to partial immersion and semi-solid medium. The chlorophyll content, stomatal index, and stomata functioning were affected by the different culture systems. The different culture systems had no effect on the survival percentage, although morphological differences were observed during acclimatization. The SETIS™ bioreactor was more efficient and resulted in better development in the plants obtained in vitro and ex vitro, which proved this system to be a useful alternative for banana micropropagation.

Bixa orellana L. (achiote) tissue culture: a review Abstract Bixa orellana L. (achiote) is a commercially important plant grown for its natural dye annatto, which is derived from the arils of seeds. Annatto, which contains the antioxidant bixin, is used in food, cosmetic, and textile industries as a natural colorant. Even though B. orellana can be propagated by seed, established tissue culture and in vitro propagation protocols exist for this plant. Organogeneses, both axillary and adventitious, as well as somatic embryogenesis, have been successfully induced from different explant sources, including cotyledons, hypocotyls, roots, stems, and leaves, and effective acclimatization protocols exist for regenerated plantlets. This present mini-review highlights the achievements in the tissue culture of achiote. In addition to using in vitro techniques for the clonal propagation of elite plants, knowledge from these protocols could be used to establish mass production systems, such as bioreactors, to facilitate year-round bixin harvest.

In vitro storage of micropropagated grapevine rootstocks at low temperature Abstract A long-term in vitro conservation protocol for five micropropagated grapevine rootstocks (3309 Couderc, 110 Richter, Vitis riparia Gloire de Montpellier, 101-14 Millardet et De Grasset, and Teleki 5C) was developed and optimized by testing the effect of culture containers (polyethylene bags versus glass test tubes), explant types (rooted versus unrooted cuttings), low-temperature regimes (1.6, 4.0, and 7.2°C), and light intensity (low versusdark). Optimal storage conditions were achieved at 4.0°C and low light intensity (3 μmol m−2 s−1), with rooted plantlets established on woody plant medium (WPM) supplemented with 15 g L−1 sucrose and 37 mg L−1cysteine in polyethylene bags. After 12 and 24 mo of optimal cold storage, the plant condition ratings (3.5 and 2.5 on a scale of 4) and survival rates (75% and 52%) were high, regardless of the rootstock genotype, as determined by visual observations and viability assays of subcultures on WPM supplemented with 15 g L−1 sucrose and 37 mg L−1 cysteine at 25°C, high light intensity (33 to 45 μmol m−2 s−1), and 16-h photoperiod. Prolonged storage at 4°C under low light intensity reduced the number of subcultures by 4- to 8-fold over 12 to 24 mo, which substantially lowered costs and labor, and provided opportunities for long-term germplasm conservation. Additionally, the use of polyethylene bags reduced storage space in the growth room and in a microprocessor temperature-controlled refrigerator, and also facilitated the safe exchange of germplasm across regulatory boundaries.

The in vitro propagation system of Citrus × latifolia (Yu. Tanaka) Yu. Tanaka (Rutaceae) affects the growth and depletion of nutriments Abstract Among citrus fruits, Citrus × latifolia (Yu. Tanaka) Yu. Tanaka stands out given its diverse uses, and there is a great demand for plants of high phytosanitary and genetic quality. In the present study, the in vitromicropropagation process of C. latifolia was studied in RITA®-type bioreactor, semisolid medium, and partial immersion with liquid culture medium. Explants that were 2 mo of age were used and apices of 0.5 cm were extracted and placed in each culture system. In all the systems, MS (Murashige and Skoog) culture medium was used, supplemented with 6-benzylaminopurine (BAP) and kinetin (KIN). After 30 d, the number of shoots, the size of the shoots, the number of leaves, and the depletion of nutrients from the culture medium were analyzed. With the data obtained, ANOVA and Tukey's mean comparison tests were performed. The RITA® bioreactors, with the addition of 40 mL of medium per explant in a 4-h-interval immersion, facilitated the production of the greatest number of shoots compared to the other culture systems, and also resulted in the highest levels in medium nutrient depletion.