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Dongmei Wei, Huimin Xu and Ruili Li

Anthers contain starch and neutral lipids, which have key roles in microspore ontogeny and gametophyte development. In this study, we observed the dynamic changes in starch and neutral lipids in the anther developmental processes of castor (Ricinus communis) by cytochemical methods. Starch grains and neutral lipids presented a regular dynamic distribution during anther development. In young anthers, some neutral lipids accumulated in sporogenous cells, whereas neutral lipids disappeared with microspore growth. At the late microspore stage, starch grains began to accumulate in microspores, and the starch content of bicellular pollen significantly increased after microspore mitosis. At anthesis, starch grains and neutral lipids accumulated in the mature pollen grains. Visible changes occurred in anther wall cells. The epidermis, middle layer, and tapetum were degenerated, and only a single layer of endothecium remained at anthesis. The dynamic variation of starch grains and neutral lipids in tapetal cells was consistent with the changes in microspores and pollen during anther development. All these findings demonstrated that tapetal cells directly interacted with the developing gametophytes. The tapetal cells play an important role in supplying nutritional substances for microspore absorption. Moreover, the endothecium protects the pollen and contributes to anther dehiscence. The results of this study provide a foundation for the further research on sexual reproduction in angiosperms.

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Li Li, Xiulan Xu, Ping Wu, Guo Zhang and Xiaobing Zhang

Twenty-four representative melon varieties and six parental cultivars were examined in this study. Among 159 pairs of simple sequence repeat (SSR) primers, 18 SSR core primers with rich polymorphic information, a large number of genotypes, and the ability to distinguish different melon varieties were selected. A total of 113 genotypes were detected among the 30 experimental materials, with an average of 6.28 genotypes for each pair of primers. The polymorphic information content was on average 0.6807, ranging from 0.5618 to 0.7885. Specific bands of the primers for the 30 experimental materials were analyzed, and by combining different primer loci, all 30 varieties were identified. Unique barcodes for molecular identity cards for the 30 experimental materials were established using the fingerprints formed with this SSR marker system. Each variety has a unique identity card that can be applied for the registration of the newly bred varieties, the protection of breeders’ rights, and the authenticity of breeds after promulgation of the new Seed Law of the People’s Republic of China.

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Li Xu, Suzhen Huang, Yulin Han and Haiyan Yuan

An improved three-stage protocol for plant regeneration via somatic embryogenesis of the horticulturally important plant Iris germanica L. was developed using shoot apex segments as explants. At the first stage of the experiment, 60% of callus was obtained from shoot apex segments of I. germanica on Murashige and Skoog’s (MS) medium supplemented with 4.52 μm 2,4-dichloropheoxyacetic acid (2,4-D) and 0.44 μm 6-benzyladenine (6-BA). When nonembryogenic calli were subcultured on MS medium with 11.31 μm 2,4-D and 0.44 μm 6-BA, maximum frequency of embryogenic callus (66.0%) was obtained. At the second stage, the treatment of 9% (w/v) sucrose resulted in the optimum somatic embryo (SE) formation (70.0%). More than 90.0% of SEs germinated with bipolar structure and regenerated into plantlets on plant growth regulator-(PGR)free MS medium during the third stage. Regenerated plantlets were successfully acclimatized in greenhouse environment with little somaclonal variation. Histological study showed that somatic embryogenesis stages were asynchronous and SEs developed from the surface and inner tissue of embryogenic calli.

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Jinhong Yuan, Man Xu, Wei Duan, Peige Fan and Shaohua Li

The responses of photosynthesis, chlorophyll fluorescence, and de-epoxidation state of the xanthophyll cycle pigments (DEPS) of micropropagated apple trees (Malus ×domestica) were investigated under whole-root water stress (WRS) and half-root water stress (HRS) induced by polyethylene glycol 6000 to simulate whole and partial root zone drying. Compared with control plants without water stress, plants under WRS and HRS exhibited reduced leaf net photosynthetic rate (Pn) and stomatal conductance (g S) with a greater reduction in WRS than in HRS plants. However, intercellular CO2 concentration (Ci) increased under WRS as water stress was prolonged, signifying a non-stomatal limitation of Pn. Regarding HRS, decreased Pn was mainly the result of a stomatal limitation explained by a relatively low Ci. Changes in photosynthesis and chlorophyll parameters indicate that severe and slight damage occurred to the photosynthetic apparatus of WRS and HRS leaves, respectively, starting at Day 3 after initiating water stress. This damage was not evident on the donor side but was expressed as a reduced capacity of the acceptor side of the photosystem II reaction centers. To prevent damage from excess light, the DEPS of WRS leaf increased. Decreased g S could explain reduced water use under an irrigation strategy of partial root zone drying in fruit trees.

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Xiaoying Li, Hongxia Xu, Jianjun Feng and Junwei Chen

Deep transcriptome sequencing allows for the acquisition of large-scale microsatellite information, and it is especially useful for genetic diversity analysis and mapping in plants without reference genome sequences. In this study, a total of 14,004 simple sequence repeats (SSRs) were mined from 10,511 unigenes screening of 63,608 nonredundant transcriptome unigenes in loquat (Eriobotrya japonica) with a frequency of 22 SSR loci distributed over 100 unigenes. Dinucleotide and trinucleotide repeat SSRs were dominant, accounting for 20.62%, and 42.1% of the total, respectively. Seventy primer pairs were designed from partial SSRs and used for polymerase chain reaction (PCR) amplification. Of these primer pairs, 54 exhibited amplification and 33 were polymorphic. The number of alleles at these loci ranged from two to 17, and the polymorphism information content values ranged from 0.24 to 0.89. We tested the transferability of 33 SSR polymorphic primer pairs in apple and pear, and the transferability rates in these two species were 90.9% and 87.9%, respectively. A high level of marker polymorphism was observed in apple [Malus ×domestica (66.7%)], whereas a low level was observed in pear [Pyrus sp. (51.5%)]. In addition, the PCR products from seven SSR primer pairs were selected for sequence analysis, and 89.2% of the fragments were found to contain SSRs. SSR motifs were conserved among loquat, apple, and pear. According to our sequencing results for real SSR loci, ≈12,490 SSR loci were present in these loquat unigenes. The cluster dendrogram showed a distinct separation into different groups for these three species, indicating that these SSR markers were useful in the evaluation of genetic relationships and diversity between and within the species of Maloideae in the Rosaceae. The results of our identified SSRs should be useful for genetic linkage map construction, quantitative trait locus mapping, and molecular marker-assisted breeding of loquat and related species.

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Feifei Li, Da Zhan, Lixin Xu, Liebao Han and Xunzhong Zhang

Heat stress is a major limiting factor for growth of cool-season perennial grass species, and mechanisms of heat tolerance have not been well understood. This study was designed to investigate antioxidant enzyme and hormone metabolism responses to heat stress in two kentucky bluegrass (Poa pratensis L.) cultivars contrasting in heat tolerance. The plants were subjected to 20/20 °C [day/night (control)] or 38/30 °C [day/night (heat stress)] for 28 days in growth chambers. Heat stress increased leaf electrolyte leakage (EL) and malondialdehyde (MDA) with heat-tolerant cultivar EverGlade exhibiting lower levels of EL and MDA relative to heat-sensitive cultivar Kenblue under heat stress. Superoxide dismutase (SOD) and catalase (CAT) activity increased and then declined during 28 days of heat stress. Peroxidase (POD) and ascorbate peroxidase (APX) activity declined and then increased during heat stress. ‘EverGlade’ had greater activities of SOD, CAT, POD, and APX relative to ‘Kenblue’ under heat stress. In addition, ‘EverGlade’ had two additional SOD isozymes and three additional POD isozymes relative to ‘Kenblue’ under heat stress. Leaf abscisic acid (ABA) increased in response to heat stress. Leaf indole-3-acetic acid (IAA) increased and then declined during heat stress. ‘OverGlade’ had higher ABA and IAA content relative to ‘Kenblue’. At the end of heat stress, leaf IAA and ABA content were 27.8% and 73% higher in ‘EverGlade’ relative to ‘Kenblue’, respectively. The results indicated that antioxidant enzymes and the hormones (ABA and IAA) were associated with kentucky bluegrass heat tolerance. Selection and use of cultivars with higher IAA and ABA content and greater antioxidant enzyme activities may improve kentucky bluegrass growth and quality under heat stress.

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Ruining Li, Wenwen Huang, Xiaoxiao Wang, Xiaoying Liu and Zhigang Xu

The objectives of this study were to determine the effects of yellow light (Y), green light (G), and two blue lights (B) at different wavelengths in conjunction with red light (R) on the growth and morphogenesis of potato plantlets in vitro. Randomized nodal explants were cut into 1.0–1.5 cm pieces and were grown under five different light conditions: fluorescent white light (FL); the combined spectra of R, Y, and B at 445 nm (R630B445Y); the combined spectra of R, G, and B at 445 nm (R630B445G); the combined spectra of R, Y, and B at 465 nm (R630B465Y); and the combined spectra of R, G, and B at 465 nm (R630B465G). Morphogenesis and physiological parameters were investigated. The results showed that R630B445Y and R630B465Y increased the fresh weight (FW), dry weight (DW), stem diameter, blade number, leaf area, specific leaf weight (SLW), and the health index of potato plantlets in vitro; root activity increased significantly; and soluble sugar, soluble protein, and starch also increased. The addition of Y to the combined spectra of R and B contributed to the growth, development, and morphogenesis more than the combined spectra of R and B with G, and B at 445 nm was more effective at promoting plant growth than was B at 465 nm.

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Haiyan Xu, Folian Li, Yuezhi Pan and Xun Gong

The investigation of hybridization processes and embryogenesis of heterozygote is an effective approach for early hybrids’ identification, which could provide reliable information for successful crossbreeding. In this study, we reported the whole hybridization processes of the direct cross and reciprocal cross between Michelia yunnanensis Franch. ex Finet et Gagnep. and Michelia crassipes Law using fluorescence microscopy after aniline blue staining, with the pollen germination on stigmas, pollen tube growth in styles, and subsequent extension into the embryo sac as well as the double fertilization processes are documented in detail. The M. yunnanensis × M. crassipes combination displayed considerable cross-compatibility, and the heterozygote embryogenesis was further observed with an approach of modified cryosectioning technique. Besides, the whole formation processes of hybrid seeds from artificial pollination to maturation were successfully observed. However, in the reciprocal cross, we found incompatibility between pollen grains of M. yunnanensis and stigmas of M. crassipes for the reason of hysteretic identification, as well as the abnormal callose deposition which belongs to the prefertilization barriers. This is the first study in which the complete and clear hybridization processes in Michelia were reported. We inferred that unilateral incompatibility of M. crassipes detected in this study may also exist in some other Michelia species. In artificial hybridization practices, we suggest some special treatments for overcoming prefertilization barrier should be taken when treating M. crassipes as the maternal parent.

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Aoxue Wang, Fanjuan Meng, Xiangyang Xu, Yong Wang and Jingfu Li

Leaf mold, caused by the fungus Cladosporium fulvum, is a serious disease of tomato. In the current study, the main physiological races of C. fulvum collected from three northeastern provinces of China were identified using a set of identification hosts. The results showed that the prevalent pathogenic physiological races were 1.2.3, 1.3, 3, 1.2.3.4, and 1.2.4. F1, F2, and BC1 tomato plants were obtained by crossing C. fulvum-resistant cultivar 03748 carrying the Cf-6 gene and susceptible cultivar 03036. Three 10-mer oligonucleotide random amplified polymorphic DNA (RAPD) primers and two simple sequence repeat (SSR) primers were selected for the further molecular marking analysis after 210 RAPD primers and 50 SSR primers were screened using the bulked segregate analysis method. The polymorphic DNA bands were amplified among parents, 10 F1 plants, 184 F2 plants including 145 resistant plants and 39 sensitive plants using three RAPD primers and two SSR primers so that three RAPD molecular markers and two SSR molecular markers linked to the Cf-6 loci were identified. Three RAPD markers were linked to the Cf-6 resistant locus separated with 8.7 cM, 20.3 cM, and 33.4 cM. Also, one RAPD codominant marker S374619/559 was found. The locations of the two SSR markers were 12.6 cM and 9.7 cM away from the Cf-6 locus. After cloning and sequencing two specific DNA fragments closely connected to the Cf-6 resistant and susceptible alleles respectively, in the RAPD codominant marker S374619/559 and one codominant sequence characterized amplified region marker S674619/559 was converted from RAPD marker S374619/559. In the RAPD marker S374619/559, the length difference of two specific fragments, 619-bp fragment and 559-bp fragment, is the result of one insertion (60 bp) in the 619-bp fragment. These markers will facilitate the selection of resistant tomato germplasm containing the Cf-6 gene and cloning of Cf-6 to breed new C. fulvum resistant tomato cultivars.

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Shaolan Yang, Changjie Xu, Bo Zhang, Xian Li and Kunsong Chen

Two complementary DNA fragments encoding expansin genes Ad-EXP1 and Ad-EXP2 were isolated from ripening kiwifruit (Actinidia deliciosa cv. Bruno) by reverse transcription–polymerase chain reaction amplification using a pair of degenerate primers. The homology between these two expansin family members was 50% in nucleotide sequence and 74% in amino acid sequence. It was revealed that Ad-EXP1 and Ad-EXP2 belong to subgroups A and B of an expansin gene family respectively. However, gene expression of these two members shared similar patterns. Both were upregulated by ethylene treatment and downregulated by acetylsalicylic acid treatment. The study suggests that members of both subgroups A and B of the expansin family are involved in kiwifruit fruit ripening.