A reduced, genotype-dependent ability to form a protective subintinal layer that serves as osmoprotective barrier to increase viability is identified as a source of recalcitrance to induction of microspore embryogenesis.

BackgroundFor in vitro culture of plant and animal cells, one of the critical steps is to adjust the initial cell density. A typical example of this is isolated microspore culture, where specific cell densities have been determined for different species. Out of these ranges, microspore growth is not induced, or is severely reduced. A similar situation occurs in many other plant and animal cell culture systems. Traditionally, researchers have used counting chambers (hemacytometers) to calculate cell densities, but little is still known about their technical advantages. In addition, much less information is available about other, alternative methods. In this work, using isolated eggplant microspore cultures and fluorescent beads (fluorospheres) as experimental systems, we performed a comprehensive comparison of six methods to calculate cell densities: (1) a Neubauer improved hemacytometer, (2) an automated cell counter, (3) a manual-counting method, and three flow cytometry methods based on (4) autofluorescence, (5) propidium iodide staining, and (6) side scattered light (SSC).ResultsOur results show that from a technical perspective, hemacytometers are the most reasonable option for cell counting, which may explain their widely spread use. Automated cell counters represent a good compromise between precision and affordability, although with limited accuracy. Finally, the methods based on flow cytometry were, by far, the best in terms of reproducibility and agreement between them, but they showed deficient accuracy and precision.ConclusionsTogether, our results show a thorough technical evaluation of each counting method, provide unambiguous arguments to decide which one is the most convenient for the particular case of each laboratory, and in general, shed light into the best way to determine cell densities for in vitro cell cultures. They may have an impact in such a practice not only in the context of microspore culture, but also in any other plant cell culture procedure, or in any process involving particle counting.Electronic supplementary materialThe online version of this article (10.1186/s13007-018-0297-4) contains supplementary material, which is available to authorized users.

Key message: 17 We report on an improved process of doubled haploid plant regeneration starting from 18 eggplant androgenic calli obtained through isolated microspore culture. known on the best in vitro conditions to promote this process. This is why in this work Production of DHs reduces the time and costs required to produce homozygous, pure 49 lines, essential for hybrid seed production. This is why there is an increasing effort to 50 extend this technology to new species and to optimize the protocols already existing for To increase the rate of normal-appearing, elongated shoots, we tested the effect of 114 different media on shoot growth and elongation (Table 1) On the other hand, some shoots were able to form roots while they were cultured in 156 elongation medium, before the stem was 2 cm long, and therefore, before being 157 transferred to rooting medium. M11 induced rooting in 12.7% of the shoots, while in the 158 other media the frequency was much lower ( Figure 4A; Table 1). These results pointed during the elongation period was relatively low (12.7%), possibly due to the fact that 165 most these shoots were not differentiated enough to be able to root spontaneously. This 166 is why we considered the addition of a rooting step (with M11) to the protocol. Around 167 71% of the elongated shoots produced in M11, M12, M13 and M14 developed roots 168 when transferred to M11, forming entire in vitro plantlets. As seen in Table 1, the 169 rooting percentages were remarkably similar, ruling out a different effect of the 170 elongation medium used in the rooting capacity of the shoots after transference to 171 rooting (basal) medium. Taken together, our short-term results (1 month) and mid-term 172 results (6 months) showed that M11 and M13 were the best performing media, and 173 while M13 produced more elongated shoots after 6 months, M11 produced more entire 174 plantlets. Besides, it appeared that the addition of an elongation step after shoot 175 initiation was beneficial not only to produce normal-appearing, elongated shoots, but 176 also to increase their rooting ability. In this work we addressed the main limiting step of DH production in eggplant through 210 microspore culture: the regeneration of entire DH plants from microspore-derived calli. Table 1, this procedure allowed us to obtain 7.6 plants every 100 220 cultivated calli, which represents a remarkable improvement (~4x) compared with the 221 2% obtained in the only previous reference of entire plant regeneration from eggplant 222 microspore-derived calli (Miyoshi 1996). We also showed that media supplemented 223 with GA 3 , and in particular M13, produced more elongated shoots, ready for rooting, 224 than any other media. However, the inability of this medium to promote root growth,

Notwithstanding the importance of eggplant in global horticulture, doubled haploid production in this species is still far from being efficient. Although acknowledged to have a role in the efficiency of androgenesis induction, factors such as the growth conditions of donor plant or the in vitro culture environment have not been deeply explored or not explored at all in eggplant, which leaves room for further improvement. In this work, we investigated the effects of different in vivo and in vitro parameters on the androgenic performance of different eggplant genotypes, including two hybrids and a DH line. The in vivo parameters included the exposure of donor plants to different temperature and light conditions and to increased levels of boron. The in vitro parameters included the use of different concentrations of NLN medium components, sucrose and growth regulators, and the suspension of microspores at different densities. Our results showed that whereas greenhouse temperature variations or boron application did not to have a positive influence, greenhouse lighting influenced their viability, thereby conditioning the embryogenic response. Changes in different sucrose, salts and hormone levels had different effects in the genotypes studied, which correlated with their genetic constitution. Finally, we determined the best microspore density, different from that previously proposed. Our work shed light on the role of different factors involved in eggplant microspore cultures, some of them not yet studied, contributing to make microspore culture a more efficient tool in eggplant breeding.

Microspore cultures generate a heterogeneous population of embryogenic structures that can be grouped into highly embryogenic structures [exine-enclosed (EE) and loose bicellular structures (LBS)] and barely embryogenic structures [compact callus (CC) and loose callus (LC) structures]. Little is known about the factors behind these different responses. In this study we performed a comparative analysis of the composition and architecture of the cell walls of each structure by confocal and quantitative electron microscopy. Each structure presented specific cell wall characteristics that defined their developmental fate. EE and LBS structures, which are responsible for most of the viable embryos, showed a specific profile with thin walls rich in arabinogalactan proteins (AGPs), highly and low methyl-esterified pectin and callose, and a callose-rich subintinal layer not necessarily thick, but with a remarkably high callose concentration. The different profiles of EE and LBS walls support the development as suspensorless and suspensor-bearing embryos, respectively. Conversely, less viable embryogenic structures (LC) presented the thickest walls and the lowest values for almost all of the studied cell wall components. These cell wall properties would be the less favorable for cell proliferation and embryo progression. High levels of highly methyl-esterified pectin are necessary for wall flexibility and growth of highly embryogenic structures. AGPs seem to play a role in cell wall stiffness, possibly due to their putative role as calcium capacitors, explaining the positive relationship between embryogenic potential and calcium levels.