Pepper (Capsicum annuum) pollen is bicellular and contains a vegetative cell and a generative cell, which divides in pollen tubes to form two sperm cells. Sperm cells of pepper were isolated using an in vivo–in vitro method. Hand-pollinated styles were first grown in vivo for several hours, then cut from their base and cultured in vitro until pollen tubes grew from the cut end. When the pollen tubes were transferred to a breaking solution, sperm cells were released from broken tubes. Viable embryo sac cells of pepper were isolated using enzymatic digestion and mechanical dissection. Isolated ovules were digested using cellulase and pectinase for 40 minutes and then transferred to an enzyme-free solution for mechanical dissection. Three cells of the egg apparatus and a central cell were released from a cut at the chalazal end of each ovule by pressing on the micropylar area of the ovule with a microneedle. Optimal isolation conditions included 11% mannitol, 0.04% CaCl2, 1% bovine serum albumin (BSA), 1% cellulase, 1% pectinase, and 0.3% pectolyase. Using this protocol, populations of pepper egg cells, synergids, and central cells were isolated.
Wei Deng, Yunling Xie, and Yilan Qiu
Zhiyong Hu, Min Zhang, Qigen Wen, Jie Wei, Hualin Yi, Xiuxin Deng, and Xianghua Xu
Seedlessness is of commercial importance in citrus (Citrus L.). Seedless ‘Ougan’ mandarin (C. suavissima) was selected from a bud sport mutation that occurred in ‘Ougan’ mandarin. We analyzed their pollen viability through KI-I2 and FDA staining, and examined the anthers of wild-type (seedy) and seedless mutant ‘Ougan’ mandarin using histological and cytochemical methods to characterize the process of pollen development. No pollen fertility was detected in this mutant. Pollen abortion in anthers of the mutant occurred at the tetrad stage of microspore development, and almost all the tetrads were abnormal. The mutant had heterogeneous microspore populations, including monads, dyads, triads, tetrads, and polyads in the same microsporangium. Pollen grain number per anther of the mutant was 21.9% less than the wild type. Morphology of mature pollen grains using SEM showed that the shape of mature pollen grains from both wild type and mutant is similar, but the microsporangia of the latter contained pollen grains of more variable sizes. At the early mature pollen grain stage, abundant starch grains and lipids appeared in the wild type's pollen, but fewer amounts were observed in the mutant. Moreover, the tapetal cells of the wild type accumulated lipids, but not those of the mutant. Results indicated that the abnormal development of the microspore led to pollen abortion in the mutant, and this could be the reason for its seedlessness. However, the genetic reasons for the aberrant tetrads are not clear and are under investigation.
Yang Hu, Chao Gao, Quanen Deng, Jie Qiu, Hongli Wei, Lu Yang, Jiajun Xie, and Desheng Liao
Petalized anther abortion is an important characteristic of male sterility in plants. The male sterile plants (HB-21) evincing petalized anther abortion previously discovered in a clone population of the Camellia oleifera cultivar Huashuo by our research group were selected as the experimental material in this study. Using plant microscopy and anatomic methods and given the correspondence between external morphology and internal structure, we studied the anatomic characteristics of petalized anther abortion (with a fertile plant as the control group) in various stages, from flower bud differentiation to anther maturity, in hopes of providing a theoretical basis for research on and applications of male sterile C. oleifera plants, a new method for the selection of male sterile C. oleifera cultivars, and improvements in the yield and quality of C. oleifera. In this study, the development of anthers in C. oleifera was divided into 14 stages. Petalized anther abortion in male sterile plants was mainly initiated in the second stage (the stage of sporogenous cells). Either the petalized upper anther parts did not form pollen sacs, or the entire anthers did not form pollen sacs. The lower parts of some anthers could form deformed pollen sacs and develop, and these anthers could be roughly divided into two types: fully and partially petalized anthers. Abnormal callose and the premature degradation of the tapetum occurred in the pollen sacs formed by partially petalized anthers during the development process, resulting in the absence of inclusions in the pollen grains formed. Small quantities of mature pollen grains withered inward from the germinal furrows, exhibiting obvious abortion characteristics. The relative in vitro germination rate of the pollen produced by the partially petalized anthers of sterile plants was 11.20%, and the relative activity of triphenyltetrazolium chloride was 3.24%, while the fully petalized anthers did not generate pollen grains. Either the petalized anthers in male sterile plants did not produce pollen, or the vitality of the small amounts of pollen produced by sterile plants was very low compared with that of fertile plants. Such male sterile plants could be used to select correct clones and have good prospects for application in production.
Wei-Ling Yuan, Shang-yong Yuan, Xiao-hui Deng, Cai-xia Gan, Lei Cui, and Qing-fang Wang
Efficient nitrogen (N) fertilizer management is crucial for ensuring the maximum economic yield and reducing the risk of environmental pollution. The objective of this study was to determine the effect of N fertilizer management on root yield and N uptake of radish in southern China by using 15N isotope tracing. A 2-year field experiment was conducted with three N rates (0, 60, and 120 kg N/ha) and two different application proportions, viz, A [50% at basal, 20% at 15 days after seeding (DAS), 30% at 30 DAS] and B (30% at basal, 20% at 15 DAS, 50% at 30 DAS) for each N rate, which were expressed as N0, N60A, N60B, N120A, and N120B, respectively. The results showed that root yields were significantly increased with N rates increasing from 0 to 120 kg N/ha. The root yields for N120A and N120B were 67.60 t·ha−1 and 72.50 t·ha−1 at harvest, 64.07% and 66.67% higher than those for the treatments of N60A and N60B, respectively. Mean radish recovery of N fertilizer ranged from 25.90% at N120A to 32.60% at N60B, and N fertilizer residual rate in the soil ranged from 11.50% at N120A to 14.90% at N60B. About 17.50% to 35.70% of total uptake of 15N derived from basal fertilizer was absorbed at seeding stage. However, 61.87% to 80.18% of total uptake of 15N derived from topdressing fertilizer absorbed at root expanding stage. Therefore, appropriate nitrogen application with increasing topdressing nitrogen amount could increase root yield of radish and the nitrogen recovery efficiency. Nitrogen fertilizer application recommended was 120 kg N/ha with 30% for basal, 20% for 15 DAS and 50% for 30 DAS in this study.
Tao Dong, Fang-cheng Bi, Yong-hong Huang, Wei-di He, Gui-ming Deng, Hui-jun Gao, Ou Sheng, Chun-yu Li, Qiao-song Yang, Gan-jun Yi, and Chun-hua Hu
An efficient biolistic transformation system of banana combined with a liquid medium selection system was developed during this study. An embryogenic cell suspension (ECS) of Musa acuminata cv. Baxi (AAA) was bombarded with a particle delivery system. After 7 days of restoring culture in liquid M2 medium, embryogenic cells were transferred to a liquid selection M2 medium supplemented with 10 μg/mL hygromycin for resistance screening. The untransformed cell clusters were inhibited or killed, and a small number of transformants proliferated in the liquid selection medium. After the 0th, first, second, and third generation of antibiotic screening, there were 0, 65, 212, and 320, respectively, vitality-resistant buds obtained from a 0.5-mL packed cell volume (PCV) of embryogenic cell suspension. The β-glucuronidase (GUS) staining, polymerase chain reaction (PCR) analysis, and Southern blot hybridization results all demonstrated a 100% positive rate of regenerated resistant seedlings. Interestingly, the number of buds obtained through third-generation screening was almost equal to that obtained from the original ECS in M2 medium without antibiotics. These results suggested that the liquid medium selection system facilitated the proliferation of a positive transgenic ECS, which significantly improved the regeneration rate of transformants. This protocol is suitable for the genetic transformation of all banana genotypes and is highly advantageous to varieties with low callusing potential.