Alteration of Flower Color in Viola cornuta cv. “Lutea Splendens” through Metabolic Engineering of Capsanthin/Capsorubin Synthesis

Flower color is an important characteristic that determines the commercial value of ornamental plants. The development of modern biotechnology methods such as genetic engineering enables the creation of new flower colors that cannot be achieved with classical methods of hybridization or mutational breeding. This is the first report on the successful Agrobacterium-mediated genetic transformation of Viola cornuta L. The hypocotyl explants of cv. “Lutea Splendens” variety with yellow flowers were transformed with A. tumefaciens carrying empty pWBVec10a vector (Llccs−) or pWBVec10a/CaMV 35S::Llccs::TNos vector (Llccs+) for capsanthin/capsorubin synthase gene (Llccs) from tiger lily (Lilium lancifolium). A comparative study of shoot multiplication, rooting ability during culture in vitro, as well as phenotypic characteristics of untransformed (control) and transgenic Llccs− and Llccs+ plants during ex vitro growth and flowering is presented. Successful integration of Llccs transgene allows the synthesis of red pigment capsanthin in petal cells that gives flowers different shades of an orange/reddish color. We demonstrate that the ectopic expression of Llccs gene in ornamental plants, such as V. cornuta “Lutea Splendens” could successfully be used to change flower color from yellow to different shades of orange.

Induction of nucellar embryogenesis in nucellar tissue of Citrus aurantifolia for their clonal multiplication

Citrus aurantifolia (lime) has been selected as explant for nucellar embryogenesis. Nucellus is a non-vascularized tissue being true-to-type same as mother plant, meristematic cells have no plasmodesmata connection, no virus can pass through nucellus, thus it seems to be a good material for production of virus freeplantlet.Putrescine at 0.25 or 0.5 mg1-1 and anapthaleneacetic acid at 0.10 mg1-1 supplemented to nutrient formulation were most effective in alleviating cotyledonary proliferation and fasciation while promoting embryo-to-embryo proliferation producing numerous whitish globular embryos were formed. For further development of globular embryos to well-differentiated cotyledonary embryos, additional presence of 2-isopentenyladenine at concentrations of 0.10 or 0.25 mg 1-1 was essential, contrary to incorporation of 0.10 or 0.25 mg 1-1 6benzylaminopurine, which promoted excessive proliferation of cotyledonary structures and their fasciation while zeatin at the same concentrations produced intermediate response. In the optimum treatment containing 0.25 mg l-1 putrescine, 0.10 mg 1-1 isopentenyladenine, 0.10 mg 1-1 indole-3-acetic acid and 100 mg l-1 malt extract, an average 10 well-developed embryos per culture were formed, besides some abnormal cotyledonary structures. Well-developed embryos measuring ca. 2 cm. in length (leaving the root) germinated 100% into plantlets, during 60 days, in the additional presence of amino acid supplement comprising, 5 mg 1-1 each of L-arginine, L-asparagine, L-histidine, L-cysteine, L-lysine and 10 mg l-1 L-glutamine. Such plantlets nurtured in a different medium attained a height of ca. 4 cm in 45 days before they were taken out for ex vitro growth. There was 100% transplant success and the plants grew normally.

Hybrid Pine (Pinus attenuata × Pinus radiata) Somatic Embryogenesis: What Do You Prefer, Mother or Nurse?

Development of hybrid pines of Pinus radiata D. Don for commercial forestry presents an opportunity to diversify the current resource of plant material. Climate change and different land uses pose challenges, making alternative species necessary to guarantee wood and non-wood products in the future. Pinus radiata var. cedrosensis × Pinus attenuata hybrid possesses different attributes, such as tolerance to drought conditions, better growth and resistance to snow damage at higher altitudes, and more importantly, different wood quality characteristics. Embryogenic cell lines were successfully initiated reciprocal hybrids using as initial explants megagametophytes, excised zygotic embryos and excised zygotic embryos plus nurse culture. However, the questions raised were: does the initiation environment affect the conversion to somatic plantlets months later? Does the mother tree or the cross have an effect on the conversion to somatic plantlets? In the present work we analysed the maturation rate, number of somatic embryos, germination rate, and the ex-vitro growth in cell lines derived from different initiation treatments, mother tree species, and crosses. Differences were not observed for in vitro parameters such as maturation and germination. However, significant differences were observed due to the mother tree species in relation with the ex-vitro growth rates observed, being higher those in which P. radiata acted as a mother. Moreover, embryogenic cell lines from these hybrids were stored at −80 °C and regenerated after one and five years.

Stimulation of ex vitro growth of Rhododendron hybrids ‘Nova Zembla’ and ‘Alfred’ by inoculation of roots with Serendipita indica

Experiments were carried out to study the consequences of inoculating Rhododendron ‘Nova Zembla’ and ‘Alfred’ microcuttings and young plants with Serendipita indica (formerly Piriformospora indica), an endophytic fungus. The inoculation at the in vitro rooting stage decreased the in vitro root and shoot quality, but after planting microplants in a greenhouse in a non-fertilised peat substrate, the plants grew 33% taller, with 10% more leaves and with more abundant roots in comparison with the non-inoculated microplants. The young plants rooted in vitro, acclimated in a greenhouse in a peat substrate and inoculated with S. indica at the time of the transplantation to the pots displayed, after 15 months, an increase in the shoot lengths by 35% and 13%, depending on the cultivar, in the leaf number by 47% and in the chlorophyll content by 31% when compared with the non-inoculated plants. The beneficial effect of S. indica on the young Rhododendron plants indicates the possibility of its practical use in the nursery production of this plant.

In vitro Plant Regeneration from Nodal Explants of Coleus forskohlii Briq. - An Important Medicinal Plant

An efficient in vitro mass propagation and promising protocol has been successfully standardized and developed for Coleus forskohlii through direct organogenesis from nodal explants. Nodal explants cultured onto MS basal medium supplemented with 4.44 μM BAP recorded the highest response and produced 24.3 ± 0.2 shoots per explant with an average shoot length 5.6 ± 0.4 cm after 30 days of culture. The in vitro shoots recorded higher response for development of rooting on half strength MS fortified with 2.46 μM IBA which produced the best response 7.8 ± 0.6 roots per in vitro shoot with an average root length of 4.3 ± 0.1 cm after 25 days. The in vitro rooted plantlets were transferred for hardening and 90% of the plantlets survived were successfully acclimatized and established in small plastic pots. This protocol recorded to be a highly repeatable, successful and rapid technique that could be utilized for the commercial mass propagation and ex situ conservation of Coleus forskohlii. It is important to note that the morphology of the in vitro plantlets of Coleus forskohlii showed a true-to-type growth habit, both in vitro and when transferred to ex vitro growth conditions. Plant Tissue Cult. & Biotech. 30(1): 143-148, 2020 (June)

Effect of Chitooligosaccharide on In vitro and Ex vitro Growth of Piper nigrum L.

Oligosaccharins: oligogalacturonic, and chitooligosaccharides are known as molecular signals to induce and regulate various genes in plants. This study was conducted to deternine the effects of chitooligosaccharide on bud formula and growth of Piper nigrum in both in vitro and ex vitro. The results showed that sterilize Piper nigrum shoots with 30% sodium hypochlorite at 10 min was the most suitable condition; appropriate culture media for bud formulation was Murashige and Skoog (MS) media supplemented 30 g/L saccharose, 7,5 g/L agar, 3 mg/L N6 – benzyl adenine (BA), culture media for growth of plantlet shoot was MS media supplemented 30 g/L sacharose, 7,5 g/L agar, 1 g/L NAA, 2 mg/L IBA and 45 ppm chitooligosaccharide. Supplementation of chitooligosaccharide at concentration of 45 ppm was optimal for the growth of Piper nigrum plantlets both in vitro and ex vitro. Present study indicated that chitooligosaccharide strongly promote the growth of Piper nigrum and recommend concentration for both in vitro and ex vitro is 45 ppm.

Height and number of shoots on the survival and development of micropropagated bamboo plantlets during pre-acclimatization

ABSTRACT During the in vitro multiplication of bamboo plantlets, it is common the formation of shoots aggregates. Once individualized, these can yield a greater number of plantlets than if planted in clusters. This study aimed to evaluate the effect of the relationship between height and number of initial shoots in micropropagated bamboo plantlets on the survival and development of plants, during the pre-acclimatization stage. Guadua aff. chaparensis shoots, after successive subcultures of in vitro multiplication, were classified into three height classes (2.5-5.0 cm; 5.1-10.0 cm; 10.1-15.0 cm) and number of aggregate shoots (one shoot per plantlet/single-stem plantlet, two and three shoots per plantlet) and pre-acclimatized in a commercial substrate composition plus washed sand. The plantlets were evaluated for survival, height, number of new shoots and roots, shoot and root fresh and dry mass. In plantlets from micropropagation, the plant height does not influence the survival rates, being acclimatized preferably at heights between 5.0 cm and 15.0 cm, with survival rates of up to 97 %. Plantlets with height starting at 5.1 cm and composed of 2 or 3 initial shoots show a greater vigor and ex vitro growth, a fact evidenced by the higher values obtained in relation to height and emission of new shoots and roots, as well as a greater fresh and dry biomass accumulation.

Cryopreservation and Acclimatization of Lycopersicon esculentum (Mill.) Genotypes

Romanian tomato (Lycopersicon esculentum Mill.) cultivars have been cryopreserved by encapsulation-dehydration and successfully acclimatized to ex vitro growth conditions. Shoot tips were excised from in vitro grown plants then precultured for 24 h in various sucrose concentrations, dehydrated up to 6 h in laminar air flow prior to direct immersion in liquid nitrogen (−196°C) for 24 h. Different parameters have been studied: the effects of osmoprotection and desiccation duration on the regrowth of cryopreserved shoot tips, the effects of various IBA concentrations on rooting and the ex vitro cclimatization of plants recovered from liquid nitrogen. The highest frequency of regrowth (72% cv. ‘Pontica’) was obtained when encapsulated explants were precultured in 0.5 M sucrose and the moisture content (fresh weight basis) of alginate beads was 23%. The highest rooting rates (58% to 77%) for all cultivars were observed for shoots grown on MS medium supplemented with 1.0 mg/l IBA. The rooted plants could be easily acclimatized ex vitro with up to 100% survival.