Comparison of the morphological structure of floral organs
Bamboo species exhibit significant variation in terms of the size and shape of their bamboo florets, as well as the spikelets, cobs, stamens, pistils, and bracts of the inflorescence structure. There were disparities in the morphology and length of flower branches between bamboo species. The flower branches of P. subsolida are densely arranged, with clustered spikelets distributed flatly, resembling the morphological characteristics observed in Bambusa oldhami[19] and D. sinicus[9]. The morphological features of Chimonobambusa utilis[20] and Sasaela kongosanensis[21] were different. In examining the morphological anatomy of floret organs, it was observed that a majority of bamboo plants had bisexual florets with either three or six stamens. P. subsolida, similar to Phyllostachys praecox[22], Chimonobambusa utilis[20], and Shibataea chinensis[12], possesses three stamens in its floret organ. The number of stamens in Dendrocalamus hamiltonii[23], Bambusa intermedia[24], B. multiplex[6], and D. sinicus[9] was six. P. subsolida possesses a pistil with a smooth and glabrous ovary, similar to the conspecific P. viridula[25], which exhibits densely ciliated and nondesquamated apices, in contrast to D. hamiltonii[23]. The six-stamen bamboo species included Dendrocalamus and Bambusa. The styles of P. subsolida are short, as indicated by the length of its stigma[26]. In bamboo plants, florets can be divided into long and short styles; therefore, P. subsolida was indicated rather than the congeneric P. viridula type because of the short styles. The mature lemma of the floret was slightly longer than the palea, while the length of the immature lemma did not differ significantly. The lemma exhibits a sharp tip and ciliated edges, with the palea positioned internally. These characteristics were similar to those of the conspecific P. viridula[25]. In P. subsolida, Bambusa oldhami, Bambusa multiplex, Bambusa intermedia, Bambusa rigida, Shibataea chinensis, Pseudosasa viridula, and Chimonobambusa utilis, the shape of the pistil was mostly a three-branched stigma; in Dendrocalamus hamiltonii and Dendrocalamus sinicus, there was an unbranched pistil, while the stigma of Arundinaria simonii contains two branches.
Bamboo plants are distinguished by their towering stature and predominantly wind mediated dispersal. In terms of pollination mechanisms, it is more difficult for wind dispersed plants to achieve widespread distribution compared to insect pollinated counterparts. Therefore, bamboo plants exhibit sporadic flowering patterns. Flowering and subsequent seed production in bamboo are rare, with limited or nonexistent seed collection even during sporadic bamboo flowering. As for the flower type, P. subsolida has continuous bloomsplants that do not decline after flowering but instead undergo asexual reproduction to renew the bamboo forest. The low seed-setting rate can be attributed to the varying types and periods of flowering observed in bamboo plants, along with potential insect infestation (in studies of P. viridula)[25].
In the field of bamboo plant reproductive biology, McClure[27] initially proposed the concept of “pseudo” spikelets and subsequently classified bamboo plant inflorescences into two categories based on the presence or absence of dormant buds located at the base of spikelets: determinate inflorescence and indeterminate inflorescence. In 1986, Geng Bojie provided a more precise definition of bamboo inflorescence as a finite true inflorescence occurring only once and an infinite false inflorescence with successive occurrences[28]. According to Zhang Zuxin's research literature, employing gene editing technology to knock out genes in the inflorescence can significantly reduce the abortion rate of maize florets, suggesting a potential association between inflorescence and this phenomenon[29]. No existing literature has been found regarding the correlation between the structure of false spikelets and inflorescences and abortion.
According to the flowering dynamics of florets in bamboo plants, upon blooming, water absorption by the pulp leads to lemma expansion. This facilitates the emergence of both male and female stamens from the lemma sheet while ensuring their simultaneous maturation. Upon completion of the powdering process, the pulp undergoes desiccation and contraction, resulting in the closure of the lemma sheet and retraction or abscission of stamens. This floral morphology corresponds to an open type. When in bloom, the lemma remains closed, with simultaneous maturation of both the pistil and stamen. This phenomenon can be classified as a closed type[17]. The most apparent distinction between the two types lies in the presence of a pulp sheet in the open type, whereas the closed type lacks such a component. In this study, P. subsolida was found to have pulp flakes and to be an open floret. In this respect, its floral features were very similar to those of Bambusa. By contrast, Dendrocalamus sinicus and D. hamiltonii did not have pulp and are of the closed type.
In this study, P. subsolida was found to exhibit infrequent blooming and a significantly low seed-setting rate within its native habitat, with no naturally occurring seedlings observed. The cytokinesis mode of the pollen mother cell was classified as the continuous type. This type was completely consistent with the spore development of M. sichuanensi[30] and was the same as that of D. sinicus[9]. However, most of the microspore tetraploids produced by D. sinicus are tetrahedral. The resulting dizygomorphic tetraploid was similar to that of B. multiplex[6], although the process of formation was not the same. The cytokinesis type of B. multiplex was simultaneous, resulting in the absence of diploid formation at the end of the first division of meiosis and direct tetraploid formation during the second division. During secondary sporulation, anther wall development involves four layers of cells: the epidermis, anther chamber wall, mesosphere, and tapetum. The anther walls of B. eutuldoides[31] and B. intermedia[24] were also fully differentiated during the secondary sporulation period, which was consistent with the development of the anther wall of P. subsolida. In contrast, the anther walls of D. sinicus[9], M. sichuanensi[30], and B. multiplex[6] were fully differentiated at the microspore mother cell stage. The anther wall of P. praecox differs from that of P. subsolida in that it lacks an inner layer, which typically consists of (from outer to inner) the epidermis, middle layer, and tapetum. The majority of mature pollen grains in P. subsolida exhibited binucleate characteristics, while a minority displayed trinucleate features. The pollen of M. sichuanensi[30] and S. chinensis[12] was similar to that of P. subsolida.
Reasons for the low seed-setting rate
The potential causes of spontaneous abortion in P. subsolida included the presence of an in-distinct or absent demarcation between tapetal cells and intermediate cells. The primary role of the tapetum was to provide nourishment and structural components for microspores [32]. During the later stages of anther development, the tape-tum undergoes deformation and releases lipids or phenols that were essential for proper anther development [33]. During the pollen grain development of P. subsolida, similar to that in most bamboo species (e.g., B. multiplex[6], B. sinospinosa[34], Shibataea chinensis[35], there was abnormal tapetum development. In contrast to D. sinicus[41], in which the tapetum is normally developed, in D. sinicus, the tapetum begins to disgroup at the stage of microspore mother cells, but it remains in its original position and does not disappear. However, in P. subsolida, although the tapetum and midlayer persist during the microspore mother cell stage, they completely disappear during meiosis and cannot provide the necessary nutrients for normal microspore development. Phyllostachys edulis[36, 37] has also been reported to have flowering (flowering in all seasons, all developmental stages) and normal fruit-bearing. Still, the anatomical structure of the female and male gametes in Phyllostachys edulis had not been described. The second point pertains to the presence of hollow microspores, which do not possess characteristics typical of pollen grains. In this study, anther shrinkage was observed during the development of P. subsolida florets; a similar phenomenon was observed in Neomi-crocalmus praini [38]and B. tuldoides[39]. Additionally, B. intermedia [40]and N. prainii [38]exhibited the contraction of other compartments, as observed in P. subsolida. However, it is hypothesized that S. chinensis exhibits limited and potentially negligible seed setting due to factors such as concealed stigma hindering effective pollination, self-pollination, and challenges associated with dioecious maturation. No instances of abnormal structural development induced abortions were observed in female or male gametophytes of D. sinicus[41] through paraffin continuous section analysis. It was worth noting that potential factors contributing to the low seed-setting rates in P. subsolida, similar to those observed in the aforementioned bamboo species, cannot be completely disregarded. Thus, there was a need for further research.
The application uses and characteristics for future research
The morphological examination and dissection of the female and male gametes in P. subsolida can fill the research gap regarding the reproductive structure of this genus and its conspecifics, while also serving as a reference for anatomical investigations into other floret structures. Simultaneously, it elucidates the reasons behind the low or even absent seed setting rate in P. subsolida, thereby explaining the physiological characteristics of bamboo plants with diminutive flowers that solely reproduce asexually. In future endeavors, advancements in bamboo plant breeding and transitioning from cuttings and transplantation to sexual reproduction can further enhance our understanding of P. subsolida's floret sterility causes.