Somatic embryogenesis in mature zygotic embryos of Picea pungens

This study developed somatic embryogenesis protocols for Picea pungens (Engelm), an important ornamental species, including initiation, proliferation, maturation, germination, and acclimation. Somatic embryogenic tissues were induced from mature zygotic embryos of five families, with a frequency of $$\ge $$ ≥ 22% for each. Embryogenic tissues (ET) from 13 clones of three families were proliferated for one week, achieving an average rate of 179.1%. The ET of 38 clones of three families were cultured in maturation medium for six weeks; 188 mature embryos on average were counted per gram ET cultured, of which $$\ge $$ ≥ 81.1% appeared normal, and each clone developed at least 28 normally matured embryos. A total of 69.9% or more of cotyledonary somatic embryos germinated normally and developed into normal emblings. The experiment of transplanting the emblings into a greenhouse had an average survival rate of 68.5%. Considerable variation among and within families during initiation and proliferation was observed, but this variation decreased in the maturation and germination. Changing the concentration of plant growth regulator of the initiation medium did not significantly change the initiation frequency. We recommend incorporating these protocols into the current Picea pungens practical programs, although further research is essential to increase efficiencies and reduce cost.

Optimization of Different Factors for Initiation of Somatic Embryogenesis in Suspension Cultures in Sandalwood (Santalum album L.)

Santalum album (L.) is a prized tropical tree species of high therapeutic and industrial importance. The wood of these naturally grown plants is extensively harvested to acquire therapeutically important metabolite santalol and be used for additional functions such as in wood statuette industries. Due to high demand, it is crucial to maintain a sufficient plant population. An easy protocol for establishing cell suspension culture initiated from the loose embryogenic callus mass of sandalwood was realized by shifting 6–8-week-old morphogenic calli acquired from the mature embryonic axis and cotyledon explant cultures in fluid media. The asynchronous embryogenic cultures were sloughed with clumps of flourishing cell clumps and embryos of various progressive phases along with diffident non-embryogenic tissues. The frequency of embryo proliferation was evidenced to determinethe expansion pace of embryogenic masses under diverse conditions. The intonation of initiation and creation of cell suspension was under the directive of the influence of exogenous plant growth regulators amended in the nutrient medium at different concentrations and combinations. Maximum relative growth rate (386%) and clumps/embryoids in elevated integers (321.44) were accomplished on MS nutrient medium fortified with 2.0 mg L−1 2,4-D in association with 0.5 mg L−1 BA and 30.0 g L−1 sucrose raised from mature embryonic axis-derived calli. Plantlet regeneration in higher frequency (84.43%) was evidenced on MS medium amended with 1.0 mg L−1 each of TDZ and GA3 in conjunction with 0.5 mg L−1 NAA and 20.0 g L−1 sucrose. Mature embryonic axis-derived calli were found to be constantly better than mature cotyledon-derived calli for raising profitable and reproducible cell suspension cultures. Regenerants displayed normal growth and morphology and were founded successfully in the external environment after hardening.

Physiological and Structural Aspects of In Vitro Somatic Embryogenesis in Abies alba Mill

Initiation of somatic embryogenesis from immature zygotic embryos, long-term maintenance of embryogenic tissue in vitro or by cryopreservation, as well as maturation, of somatic embryos of Abies alba Mill. are reported in this study. For the initiation of embryogenic tissues, a DCR medium containing different types of cytokinins (1 mg.L−1) were tested. During three consecutive years, 61 cell lines were initiated out of 1308 explants, with initiation frequencies ranging between 0.83 and 13.33%. The type of cytokinin had no profound effect on the initiation frequency within one given year. Microscopic observations revealed presence of bipolar somatic embryos in all initiated embryogenic tissues. Besides the typical bipolar somatic embryos, huge polyembryonal complexes, as well as “twin” embryos, were observed. Maturation of somatic embryos occurred on a DCR medium supplemented by abscisic acid (10 mg.L−1), polyethylene glycol (PEG-4000, 7.5%) and 3% maltose. The maturation capacity was cell-line dependent. All of the four tested cell lines produced cotyledonary somatic embryos, though at different quantities, of 16 to 252 per g of fresh weight. After germination, seedlings developed, but their further growth soon stopped after the formation of a resting bud. Altogether, seven cell lines were cryopreserved, using the slow-freezing technique. After rewarming, all tested cell lines showed regrowth rates between 81.8 and 100%.

Development-Related miRNA Expression and Target Regulation during Staggered In Vitro Plant Regeneration of Tuxpeño VS-535 Maize Cultivar

In vitro plant regeneration addresses basic questions of molecular reprogramming in the absence of embryonic positional cues. The process is highly dependent on the genotype and explant characteristics. However, the regulatory mechanisms operating during organ differentiation from in vitro cultures remain largely unknown. Recently, miRNAs have emerged as key regulators during embryogenic callus induction, plant differentiation, auxin responses and totipotency. Here, we explored how development-related miRNA switches the impact on their target regulation depending on physiological and molecular events taking place during maize Tuxpeño VS-535 in vitro plant regeneration. Three callus types with distinctive regeneration potential were characterized by microscopy and histological preparations. The embryogenic calli (EC) showed higher miRNA levels than non-embryogenic tissues (NEC). An inverse correlation for miR160 and miR166 targets was found during EC callus induction, whereas miR156, miR164 and miR394 displayed similar to their targets RNA accumulation levels. Most miRNA accumulation switches took place early at regenerative spots coincident with shoot apical meristem (SAM) establishment, whereas miR156, miR160 and miR166 increased at further differentiation stages. Our data uncover particular miRNA-mediated regulation operating for maize embryogenic tissues, supporting their regulatory role in early SAM establishment and basipetala growth during the in vitro regeneration process.

Tissue regeneration of Abies embryogenic cell lines after 1 year storage in liquid nitrogen

Embryogenic tissues of hybrid firs (