Search
Submit Manuscript
Advanced Search
MAP  >  Tropical Plants
2023 Volume 2
Article Contents
Abstract
Introduction
Materials and methods
Plant materials and low temperature treatments
Ion leakage rate determination
Analysis of cold-induced transcriptomic changes in A. thaliana and C. equisetifolia
GO enrichment analysis of cold-induced genes
Cis-regulatory binding motifs analysis of CBFs
qPCR validation of cold-related genes
Transgenic plant generation
Results
Differences in cold resistance of A. thaliana and C. equisetifolia
Differential expression patterns induced by chilling stress
Cold-induced genes GO enrichment assay
Comparison of C. equisetifolia and A. thaliana GO enriched cold-response entry genes
Confirmation of the expression profiles of selected cold response genes by qPCR in C. equisetifolia
Functional characterization of CBFs in C. equisetifolia
CBF binding motif analysis in promoter region of cold-induced genes in C. equisetifolia
The expression analysis of CBF upstream genes
Analysis on the cold intolerant mechanism in tropical and subtropical plants
Discussion
Acknowledgments
Conflict of interest
Rights and permissions
References
About this article
Next Previous
ARTICLE   Open Access    

Artificial pollination can improve fruit set and quality in the ice cream tree (Casimiroa edulis)

  • 1.

    Sanya Nanfan Research Institute of Hainan University, Hainan Yazhou Bay Seed Laboratory, Sanya 572025, China

  • 2.

    Collaborative Innovation Center of Nanfan and High-Efficiency Tropical Agriculture, School of Tropical Crops, Hainan University, Haikou 570228, China

  • 3.

    School of Health, Medical & Applied Sciences, CQ University Australia, Bruce Hwy, North Rockhampton, QLD 4701, Australia

  • 4.

    Hainan Shengda Modern Agriculture Development Co., Ltd., Qionghai 571425, China

More Information
  • Ice cream fruit (Casimiroa edulis La Llave) is a member of the Rutaceae family native to South America, which has recently been introduced to China. In order to improve the yield of ice cream fruit, we carried out a hybridization experiment with paired pollination. Pollen was collected in the morning and carefully stored, before it was applied to the stigmata of sprouted flowers, using a cotton swab or tweezers. This artificial pollination is repeated at different times of the day. This was continued until the stamens fall off the flowers, the stigmata withered, and the fruit began to develop. Fruit yield and quality were recorded as the principal metrics of the success of each hybrid. At the Hainan Tropical Fruit Window Agricultural Company, nine different ice cream fruit varieties were selected and divided into three groups (abundant pollen, little pollen and no pollen) according to the amount of pollen that each variety naturally produced. Four experimental conditions were investigated: 1. No artificial pollination (natural self-pollination); 2. Artificial self-pollination; 3. Cross-pollination (no emasculation); 4. Cross-pollination (with emasculation). The results indicated that the fruit from cultivars producing abundant pollen generally had poor quality, smaller size, average sweetness, and a medium rate of natural fruit set. In contrast, cultivars producing little pollen showed better fruit quality, higher fruit fertility and increased success rates for artificial pollination. The N2 ice cream fruit cultivar showed the best characteristics as the paternal line (pollen source), while the 4P cultivar showed the best characteristics as the maternal line (flower source). The New varieties (N2 × 4P) performs well in fruit quality, fruit size, fruit sweetness and fruit setting rate. Consequently, this cross-pollinated cultivar is recommended for agricultural production and planting.

  • Introduction

    Low temperature is one of the main factors restricting plant growth, development and geographic distribution. Cold stress is categorized as chilling (0−15 °C), generally suffered by tropical and subtropical plants, and freezing (< 0 °C), experienced by temperate plants[1]. The adaptability of plants to chilling and freezing is termed as chilling resistance and freezing resistance, respectively.

    To adapt to cold climates, plants have developed a set of complex mechanisms[2]. Lots of temperate plants increase their freezing tolerance through cold acclimation (CA). The molecular mechanism of cold acclimation in Arabidopsis has been well studied, and a relatively clear cold regulatory network has been established. The CBF/DREB1 (C-REPEAT BINDING FACTOR/DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 1)-dependent transcriptional modulation pathway takes indispensable parts in plant response to both chilling and freezing[3]. In the CBF-dependent pathway, the expression of CBF genes are rapidly and significantly up-regulated by low temperature, and the corresponding CBF proteins activate the cold-regulated (COR) genes' expression through specific binding to the C-repeat/DRE cis-elements (G/ACCGAC) motif contained in the promoters of COR genes, resulting in the improvement of cold acclimation and freezing tolerance[4−6]. Moreover, the deletion of the C-repeat/DRE regulatory motif of AtCBF2 decreases the cold tolerance of plants[7].

    CBFs function as a molecular switch under low temperature stress and is highly conserved in different higher plants. The transgenic plants with the overexpressing of endogenous CBFs represent distinctly improved tolerance to freezing[8−12]. In Arabidopsis, CBF1 (DREB1B), CBF2 (DREB1C), and CBF3 (DREB1A) are highly similar in sequence and redundant in function. The triple mutants of CBF1 CBF2 CBF3 (cbf1 cbf2 cbf3) fail to respond to chilling stress and are hypersensitive to freezing stress after cold acclimation[13]. Previous research reported that the expression of CBFs is directly modulated by some other transcription factors, such as Inducer of CBF expression 1 (ICE1)[14−16], Calmodulin-binding Transcription Activators (CAMTAs)[17,18], MYB15[19], Ethylene-Insensitive 3 (EIN3), Brassinazole-Resistant 1/BRI1-EMS-SUPPRESSOR1 (BZR1/BES1)[20−22], Phytochrome-Interacting Factors 3/4/7 (PIF3/4/7)[23,24], and Circadian Clock-Associated 1/Late Hypocotyl (CCA1/LHY)[25,26]. In addition, some genes independent CBF pathway have also been identified to be involved in cold response, such as HY5 (ELONGATED HYPOCOTYL 5)[27] and HSFA1[28].

    Plants from temperate-zones exhibit a strong resistence to low temperature conditions, whereas plants originating from the tropics and subtropics are generally sensitive to the chilling temperatures, and undergo irreversible damage[29−31]. Many studies have investigated the physiological and biochemical response of tropical and subtropical plants under cold stress, but, besides identifying some cold responsive genes, limited systematic research has been conducted to understand the reasons for the tropical and subtropical plants sensitivity to cold conditions. For example, it has been shown that the CBF-dependent pathway could perform its functions in rubber tree (Hevea brasiliensis)[32] and some genes involved in cold adaptability, such as HbICE1[33], HbCOR47[34] and HbEBP1[35] have been characterized. Compared to Cavendish (a cultivar of banana), the stonger cold resistence of another specie Dajiao could be due to the quick activation and induction of ICE1 and MYBS3[36]. In Moso bamboo, PeDREB1 is highly up-regulated by cold and PheMYB4-1 also mediated the response of bamboo to cold[37,38]. Further transcriptome analysis under different cold conditions in Moso bamboo revealed that most of the genes involved in cold response are late-responsive ones[39]. So, it is very important to improve the cold resistence of tropical and subtropical economic tree species through elucidating the underlying basis of intolerance to cold stress at the molecular level.

    Casuarina equisetifolia (C. equisetifolia) is a drought-, salinity-, and barren-tolerant tree species, and native to southeastern Australia[40]. It is a typical tropical plant with poor cold tolerance with 22.1−26.9 °C being the suitable temperature for its growth[41]. In this study, C. equisetifolia was used as a typical example of tropical and subtropical tree. We compared transcriptome of C. equisetifolia to that of A. thaliana, in which a clear view of the cold signal network has been investigated, under cold stress for different treatment times to identify the factors causing the cold intolerance in C. equisetifolia.

    Materials and methods

    Plant materials and low temperature treatments

    The seeds of C. equisetifolia were sown in the pot and grown in greenhouses for two months and then the chilling treatment was carried out. A. thaliana seeds of wild-type (Col), mutants and transgenic plants were placed on Murashige and Skoog (MS) medium for 7 d at 25 °C with 16 h/8 h light/dark cycle and then transferred into the greenhouse. After two weeks, the seedlings of A. thaliana were exposed to low temperatures.

    The seedlings of both C. equisetifolia and A. thaliana were firstly treated at 4 °C for 7 d for survival rate analysis and at 4 °C for 4 d for ion leakage experiment in a climate-controlled chamber, respectively, and then transferred to −8 °C for 6 h. Subsequently, the treated seedlings were recovered at 4 °C for 12 h and then moved to greenhouses for 7 d. Finally, the survival and ion leakage rates were calculated.

    Ion leakage rate determination

    Leaves (100−200 mg) were detached from cold-treated and untreated A. thaliana and C. equisetifolia, respectively, and were washed three times using deionized water. The samples were then placed on the clean glass chambers with 10 mL deionized water and were shaken at room temperature for 1 h to mix. The electrical conductivity (C1) of the solution was detected using a conductivity meter (DDS-307W, Gallop). Subsequently, the chambers were boiled in a water bath for 20 min, followed cooling for 1 h. After that, the electrical conductivity (C2) of the solution in each tested chamber was surveyed. The relative electrolyte leakage (REL) was obtained based on the formula REL = C1/C2 × 100%. The experiments were performed three times and the data were represented as the means ± standard deviation (SD) of three independently biological replicates.

    Analysis of cold-induced transcriptomic changes in A. thaliana and C. equisetifolia

    In order to identify the differential transcriptomic changes between A. thaliana and C. equisetifolia under cold stress, 21-day old A. thaliana and two-month old C. equisetifolia seedlings were used for low temperature treatments, which were 4 °C for 0, 2, and 24 h for A. thaliana and 0, 10 min, 2 h, 24 h, and 168 h for C. equisetifolia. Total RNA was isolated from the treated seedlings and evaluated by NanoDrop ND-1000. Poly (A)-RNA was enriched for constructing cDNA library and sequencing by Illumina NovaSeq 6000 (2 × 150 bp paired-end reads). Each time point contained three replicates.

    In order to analyze the RNA-seq dataset, we designed a pipeline. In brief, we aligned the clean reads of the samples to the corresponding genome and reference genes using hisat2-2.1.0[42] with the parameters (--min-intronlen 20 --max-intronlen 5,000 --rna-strandness RF). Gene expression in the normal and after the onset of cold treatment was determined using StringTie v2.0.3[43]. Trimmed Means of M values (TMM) values were used to represent gene expression value. The cold-induced genes were defined with more than two-fold expression under cold stress relative to that under normal conditions.

    GO enrichment analysis of cold-induced genes

    The GO term annotations of A. thaliana genes were directly downloaded from the website The Arabidopsis Information Resource (www.arabidopsis.org) (TAIR10)[44]. As reported in previous research[45,46], the protein coding genes of C. equisetifolia were annotated with the highest similarity to A. thaliana proteins through BLASTP searches (E-value < 1e–5). The GO enrichment analysis of cold-induced genes was performed through the OmicShare tools (www.omicshare.com/tools).

    Cis-regulatory binding motifs analysis of CBFs

    To explore the difference of CBF-CORs-mediated cold signaling pathway between A. thaliana and C. equisetifolia, the following pipeline was developed. First, we collected the well-known CBF regulons with cold-elevated expression in A. thaliana from previous report[47]. Next, we identified the orthologous of those CBF regulons in C. equisetifolia using BLASTP program (E-value < 1e–10). Then, we extracted 1- and 2-Kb upstream promoter sequences of the regulons in A. thaliana and C. equisetifolia from the corresponding genomes, respectively. Besides, the published A. thaliana CBFs cis-regulatory motifs (G/ACCGAC) from early research were organized[47,48]. Using the modules as query, those promoters were searched by FIMO[49]. Finally, the CBF binding sites in 1- and 2-Kb upstream promoter sequences of CBF regulons were identified.

    qPCR validation of cold-related genes

    This study utilized quantitative reverse transcription PCR (qPCR) following the methodology described by Cheng et al.[35]. The primers of genes for quantification were designed using Primer 5 software and listed in Supplemental Table S1. The primers were synthesized by Hangzhou Youkang Biotechnology Co., Ltd (Hangzhou, China). qPCR was performed using the ChamQTM SYBR Color qPCR Master on a Bio-Rad Iq5 RT-PCR instrument, following the experimental protocols outlined in the instruction manual. The data was analyzed using 2−ΔΔCᴛ method.

    Transgenic plant generation

    The full-length coding sequences (CDS) of CeqCBF genes (CeqCBF1 and CeqCBF3) were amplified and cloned into the pCAMBIA1300-35S::GFP vector between the Sma I and Xba I sites, termed as 35S::CeqCBF1OE/cbfs and 35S::CeqCBF3OE/cbfs. The recombinant plasmid was transformed into Agrobacterium tumefaciens EHA105. The transgenic lines were generated through Agrobacterium-mediated floral-dipping method and screened by Hygromycin B. The seeds from T3 homozygous transgenic plants were used for phenotype detection under low temperature conditions. The primers used are listed in Supplemental Table S2.

    Results

    Differences in cold resistance of A. thaliana and C. equisetifolia

    The molecular regulation mechanism of A. thaliana in response to cold stress is well documented. To investigate the cold stress response of C. equisetifolia, we compared the differences in cold response between A. thaliana and C. equisetifolia, and determined the impact of cold acclimation on improving cold resistance in these plant species. The morphological performance of both A. thaliana and C. equisetifolia with (4 °C for 7 d and then treated at −8 °C for 6 h) and without (directly exposed to −8 °C) cold acclimation was significantly different. Compared to the cold-acclimated (CA) counterparts, plants (both A. thaliana and C. equisetifolia) non-acclimated (NA) showed typical cold injury symptoms like desiccated foliage and eventually almost all plants died (Fig. 1a, b). Moreover, C. equisetifolia displayed higher cold sensitivity than A. thaliana (Fig. 1b). The survival rate of CA plants (both A. thaliana and C. equisetifolia) was higher than that of NA plants. In detail, the survival rate of A. thaliana achievd 95.8% after cold acclimation for 7 d, whereas the survival rate of C. equisetifolia was about 4.93% (Fig. 1c), indicating that cold acclimation could improve the cold resistence of plants and C. equisetifolia had a weaker cold acclimation capacity than A. thaliana.

    Figure 1.  Analysis of cold resistance of A. thaliana and C. equisetifolia. (a), (b) Freezing phenotypes (c) survival rates, and (d) ion leakage, of three-week-old wild type A. thaliana and two-month-old C. equisetifolia. The seedlings were grown under a 16-h-white light/8-h-dark photoperiod (LD) at 25 °C and either directly exposed to freezing treatment (non-acclimated, NA), or treated at 4 °C for 4 d or 7 d before freezing treatment (cold-acclimated, CA). Representative photographs were taken after a 3-d recovery period, survival rates were calculated, and the ion leakage was measured. Data are presented as the mean ± standard deviation of mean of three biological replicates. Asterisks represent significant differences compared to the non-acclimated plant. ** p < 0.01.
    DownLoad: Full-Size Img PowerPoint

    The relative electrolyte leakage is a vital indicator of membrane permeability. So, the electrolyte leakage assay was carried out in both plants with and without cold acclimation based on the method reported by Jiang et al.[50]. The data showed that the electrolyte leakage of CA plants was dramatically lower than that of the NA counterparts (Fig. 1d). Moreover, the influence of cold acclimation on electrolyte leakage was more obviously in A. thaliana (dropped from 74.2% to 24.5%) than in C. equisetifolia (dropped from 62.9% to 45.8%) (Fig. 1d).

    Differential expression patterns induced by chilling stress

    To further investigate the transcriptional response of C. equisetifolia under cold stress, C. equisetifolia and A. thaliana wild-type (Col) seedlings were exposed to a cold treatment at 4 °C for different time periods (0, 10 min, 2 h, 24 h, 168 h for C. equisetifolia, and 0, 2 h, 24 h for A. thaliana). Leaves from the treated seedlings were harvested at indicated time points and 24 cDNA libraries (15 for C. equisetifolia and nine for A. thaliana) were prepared which were subjected to paired-end sequencing. According to Pearson Correlation Coefficient (PCC), the sample replicates were clustered closely and independently, illustrating that the results were highly reproducible with good quality (Supplemental Fig. S1).

    To identify the genes up-regulated by cold stress, the expression of a particular gene was considered as cold-induced with log2 (fold-change) ≥ 1 (p value < 0.01) in comparison with control. The genes with read counts < 5 in each sample were removed.

    According to the Venn diagram, a total of 3,085 (1,376 at 2 h, 2,416 at 24 h) cold-induced genes were identified in A. thaliana, whereas a total of 3081 genes were up-regulated by chilling in C. equisetifolia (48 at 10 min, 189 at 2 h, 728 at 24 h, and 2,764 at 168 h) (Fig. 2a). Further, we found that there was a considerable difference in the number of cold-induced genes at each treatment time point between C. equisetifolia and A. thaliana, such as 189 vs 1,376 at 2 h, 728 vs 2,416 at 24 h, while the number of cold-induced genes at 168 h in C. equisetifolia was close to the number at 24 h in A. thaliana (Fig. 2b), displaying an obvious delay in cold response in C. equisetifolia. Moreover, expression profiles and clustering of cold-induced genes showed that numerous genes in A. thaliana was significantly induced by cold at 2 and 24 h after the start of cold treatment, while only fewer genes were up-regulated in C. equisetifolia at these time points, indicating that C. equisetifolia may require a more prolonged cold treatment than A. thaliana for transcriptome response (Fig. 2c).

    Figure 2.  Comparison of the number of cold-induced genes at different time points in A. thaliana and C. equisetifolia. (a) Venn diagram illustrating the cold-induced genes at 10 min, 2, 24 and 168 h (C. equisetifolia) and 2 h, 24 h (A. thaliana) after chilling treatment. (b) The number of cold-induced genes between the different plants and time points. (c) Expression profiles and clustering of 3,081 cold-induced genes in C. equisetifolia and 3,085 cold-induced genes in A. thaliana at different times after cold acclimation.
    DownLoad: Full-Size Img PowerPoint

    Cold-induced genes GO enrichment assay

    GO enrichment analysis was carried out to classify the putative functions of cold induced genes. The top 15 highly enriched functional groups are shown in Fig. 3. For A. thaliana, 15 functional groups and 11 functional groups separately exhibited significant enrichment within the biological processes category during the 2 h and 24 h of cold stress. These enriched groups included response to abiotic stimulus, cold, temperature stimulus, water deprivation and so on (Fig. 3a).

    Figure 3.  GO enrichment analysis of cold-induced genes at different time points in A. thaliana and C. equisetifolia under 4 °C cold treatment. (a) A. thaliana, (b) C. equisetifolia.
    DownLoad: Full-Size Img PowerPoint

    For C. equisetifolia, 13 functional groups were significantly enriched at the 10 min time point within the biological processes category, and two functional groups were enriched within the molecular process. For the 2 h time point, enriched biological processes included response to endogenous stimulus, chemical, and cold, etc., while the enriched molecular processes included beta-amylase activity and transcription factor activity. At the 24 h time point, 14 groups were enriched within the biological processes category. And at 168 h after the initiation of chilling, the GO enrichment analysis revealed that 11 functional groups were enriched in the category of biological processes (Fig. 3b). It is worth mentioning that, with the exception of the 10 min time point in C. equisetifolia, biological process 'response to cold' was represented in both C. equisetifolia and A. thaliana at all cold treatment time points, indicating the induction of cold response at the transcriptional level.

    To gain further insights, the differences in GO entries between A. thaliana and C. equisetifolia at 2 h and at 24 h treatment time points were analyzed, respectively. The results showed that GO entries related to stress response, such as hormone, abiotic stimulus, cold, and water deprivation were dramatically enriched in A. thaliana at 2 h, whereas in C. equisetifolia at 24 h (Supplemental Fig. S2a). Furthermore, at each time point, the top 200 enriched GO entries were examined and found that the two species shared 115 GO entries at 2 h of A. thaliana and 24 h of C. equisetifolia, as well as at 24 h of A. thaliana and 168 h of C. equisetifolia (Supplemental Fig. S2b). These results implied that though the cold stress response of C. equisetifolia was comparatively delayed, the overall biological enrichment classes are similar to A. thaliana.

    Comparison of C. equisetifolia and A. thaliana GO enriched cold-response entry genes

    As plant cold stress tolerance is a polygenic controlled trait[51], an array of genes and proteins play vital roles in cold tolerance[52−55]. To analyze the cold response pathway in C. equisetifolia, we extracted cold induced genes from A. thaliana transcriptome and then identified the homologous genes in C. equisetifolia using A. thaliana cold-induced genes as the query. A total of 139 and 143 genes were identified in C. equisetifolia and A. thaliana, respectively, in which 87 were significantly up-regulated by low temperature both in A. thaliana and in C. equisetifolia (Fig. 4a). However, the expression of most of them in C. equisetifolia was dramatically increased at 168 h and obviously later than that in A. thaliana, which were induced at 24 h (Fig. 4a), inferring that their delayed expression may influence the cold tolerance of C. equisetifolia. In addition, 52 genes had no change in transcription level under cold conditions in C. equisetifolia but up-regulated A. thaliana (Fig. 4b). Some of these genes have been reported to play notable roles in response to cold, such as RD29A/B[56], COR413PM1[57,58], KIN1/KIN2 (ABA-inducible protein-coding), SUS1, DAG2, and LOS2[47]. Besides, four genes namely STMP2 (Salt Tolerance-Associated Membrane Protein 2), HTT5 (HEAT-INDUCED TAS1 TARGET 5), COR15A (Cold regulated 15A), and COR15B were absent in the transcriptome of C. equisetifolia (Fig. 4c).

    Figure 4.  Comparison of the 'cold response entries' in A. thaliana and C. equisetifolia. (a) Conserved cold-induced genes in A. thaliana and C. equisetifolia. (b) Unresponsive to cold stress/ weakly cold-induced (FC < 2) genes in C. equisetifolia. (c) Gene absent in C. equisetifolia.
    DownLoad: Full-Size Img PowerPoint

    Based on the above analysis, we postulated three reasons for relative cold sensitivity of C. equisetifolia. First, compared with A. thaliana, the expression of cold responsive genes was delayed. Second, partial key cold-response genes were not up-regulated under cold stress, and last but not least, several functional cold responsive genes reported in A. thaliana were absent in C. equisetifolia.

    Confirmation of the expression profiles of selected cold response genes by qPCR in C. equisetifolia

    To confirm the expression profiles of cold responsive genes in C. equisetifolia, the transcriptional level of some genes was analyzed through qPCR. We selected two genes with the similar expression pattern to A. thaliana (CIPK7, GOLS3), seven genes with delayed cold-induced expression (DEAR1, BBX29, CBL1, MPK3, RD29A, WRKY33, CBF1), and seven genes with no change in expression (ADH1, DGK2, KIN1, LOS2, COR413-PM1, ELF3, SUS1) in C. equisetifolia under cold stress. The qPCR results revealed that the expression of CIPK7 and GOLS3 was markedly induced under cold conditions at 2 h and 24 h in C. equisetifolia, which aligned with the transcriptome data of A. thaliana (Fig. 5). On the other hand, the expression of DEAR1, BBX29, CBL1, MPK3, RD29A, WRKY33, and CBF1 in C. equisetifolia was prominently up-regulated at 168 h, whereas in A. thaliana, the up-regulation was observed a significant delay at 24 h (Fig. 5). Furthermore, the expression of ADH1, DGK2, KIN1, LOS2, COR413-PM1, ELF3, and SUS1 was not increased by cold stress in both species, and some of these genes were even down-regulated (Fig. 5). In addition, the expression of ADH1, DGK2, KIN1, LOS2, COR413-PM1, ELF3, and SUS1 was not induced by cold stress, instead a few of these were down-regulated (Fig. 5). These findings demonstrated that the qPCR results for the selected genes in C. equisetifolia were consistent with the transcriptome data of both A. thaliana and C. equisetifolia.

    Figure 5.  Expression of cold-response genes in C. equisetifolia, containing two similar expression pattern genes (CIPK7, GOLS3), seven delayed genes (DEAR1, BBX29, CBL, MPK3, RD29A, WRKY33, CBF1), four weakly responsive genes (ADH1, DGK2, KIN1, LOS2) and three non-responsive genes (COR413-PM1, ELF3, SUS1). CIPK7, CBL-interacting protein kinase 7; GOLS3, galactinol synthase 3; DEAR1, cooperatively regulated by ethylene and jasmonate 1; BBX29, B-box type zinc finger family protein; CBL, calcineurin B-like protein 1; MPK3, mitogen-activated protein kinase 3; RD29A, low-temperature-responsive protein 78 (LTI78); WRKY33, WRKY DNA-binding protein 33; ADH1, alcohol dehydrogenase 1; DGK2, diacylglycerol kinase 2; KIN1, stress-responsive protein (KIN1)/stress-induced protein (KIN1); LOS2, involved in light-dependent cold tolerance and encodes an enolase; COR413-PM1, cold regulated 413 plasma membrane1; ELF3, hydroxyproline-rich glycoprotein family protein; SUS1, encodes a protein with sucrose synthase activity. The values are the mean ± standard deviation of three biological replicates. Relative expression in untreated plants (0 h) was set to 1. *p < 0.05, **p < 0.01.
    DownLoad: Full-Size Img PowerPoint

    Functional characterization of CBFs in C. equisetifolia

    It is well known that the C-repeat binding factor (CBF) plays a predominant role in cold acclimation to gain the maximum cold resistence in plants[5,59]. We identified the Arabidopsis homologous of CBFs in C. equisetifolia and descovered that the transcriptional level of CeqCBF1 was up-regulated by low temperature (Fig. 5). To verify whether cold intolerance of C. equisetifolia was associated with CBFs, we identified CBF family in C. equisetifolia. There were eight CBF homologous proteins in the transcriptome of C. equisetifolia by BLASP, and we constructed the phylogenetic tree to determine the evolutionary relationship of C. equisetifolia CBFs (Fig. 6a). Further, the conserved structure domains of CBFs were aligned. The results showed that signature1 and AP2 domain were highly conserved, but an amino acid R was absent from signature2 in C. equisetifolia and the first amino acid D in signature2 in A. thaliana was changed into E (ESAW-) in C. equisetifolia (Fig. 6a), inferring that it may impact CBFs' function in cold response pathway. The chromosome localization data showed that A. thaliana CBF1, CBF2, and CBF3 are arranged in series on chromosome 4 (Fig. 6b), which was consistent with the previous research[60,61]. The analysis of CeqCBF genes localization on the chromosomes in C. equisetifolia revealed that four out of eight genes were found in tandem arrangements (Fig. 6c).

    Figure 6.  CBF in C. equisetifolia was involved in cold regulation. (a) Alignment of AP2/ERE domain amino acid sequences between A. thaliana and C. equisetifolia. (b), (c) Chromosome positions of CBF genes. (d) Freezing phenotypes, (e) survival rates and (f) the expression of CeqCBF1 in A. thaliana cbf triple mutants and transgenic lines with overexpressing of CeqCBF1. Two-week-old plants grown on MS plates at 22 °C were treated at −6 °C for 1 h after cold acclimation at 4 °C for 3 d. Asterisks indicate significant differences compared to the cbfs mutant plants. **p < 0.01.
    DownLoad: Full-Size Img PowerPoint

    To study the function of CeqCBFs in response to cold stress, the recombination plasmids were separately constructed with CeqCBF1 (Casgl53S05669) (exhibiting high homology to AtCBF1/AtCBF2/AtCBF3) and CeqCBF3 (Casgl344S25208) under 35S promoter and then transferred into Arabidopsis cbfs triple mutants (cbf1cbf2cbf3) by floral dipping method. The results of phenotype analysis showed that the transgenic lines with CeqCBF1 overexpression (35s::CeqCBF1OE-25/cbfs, 35s::CeqCBF1OE-27/cbfs, 35s::CeqCBF1OE-28/cbfs) and CeqCBF3 overexpression (35s::CeqCBF3OE-20/cbfs, 35s::CeqCBF3OE-27/cbfs, 35s::CeqCBF3OE-40/cbfs) in the cbfs triple mutants background could restore the sensitivity of cbfs triple mutants to cold stress to a certain extent and display the increased cold tolerance, respectively (Fig. 6d, Supplemental Fig. S3a). The survival rate of transgenic lines was obviously higher than that of cbfs triple mutants (Fig. 6e, Supplemental Fig. S3b). These results indicated that CeqCBF1 and CeqCBF3 dramatically enhance the cold tolerance of transgenic plants, though signature2 domain in CeqCBFs was different from that in AtCBFs.

    CBF binding motif analysis in promoter region of cold-induced genes in C. equisetifolia

    CBFs, vital transcription factors in plant cold acclimation, promote the expression of a subset of cold responsive (COR) genes by specific binding to a CRT/DRE (G/ACCGAC) element in their promoters[4,5]. We identified the downstream target genes of CBFs from the transcriptome of both A. thaliana and C. equisetifolia, and subsequently analyzed the binding motif contained in COR genes' promoter region.

    For A. thaliana, we identified 91 downstream target genes of CBFs in transcriptome[47], and most of them were highly induced by cold stress. Taken them as query, the same number homologous genes in C. equisetifolia were also identified. According to the transcriptome data, only half of the homologous genes in C. equisetifolia were up-regulated under low temperature condition, and a majority of the up-regulated genes exhibited a delayed response (Fig. 7). To investigate the CBF‐binding motif, the sequence of promoter located 1,000−2,000 bp upstream of ATG were analyzed by using FIMO[49]. In the 2,000 bp region, all genes contained CRT/DRE elements in A. thaliana, but only 58 of 91 contained CRT/DRE core motifs in C. equisetifolia. Moreover, the number of CRT/DRE binding motifs in the promoter of COR genes in C. equisetifolia was less than that in A. thaliana (Fig. 7), suggesting that the reduced number of CRT/DRE elements in the promoter region of C. equisetifolia may be associated with the delayed induction of COR genes in response to low temperature. Additionally, only half of the homologous genes in C. equisetifolia were up-regulated under cold treatment further supported the potential link between the decrease in CRT/DRE motifs and the reduced number of up-regulated genes in the downstream targets of CBFs in C. equisetifolia.

    Figure 7.  Heatmap and CRT/DRE element analysis of downstream cold response genes directly regulated by CBF in A. thaliana and C. equisetifolia.
    DownLoad: Full-Size Img PowerPoint

    To prove this hypothesis, the expression of 13 genes without CRT/DRE element were detected in C. equisetifolia under cold stress. Of the genes, only two (GOLS1 and NRT 1.7) genes were significantly increased at the 24 h time point under low temperature treatment, which was similar to that in A. thaliana (Fig. 8). The expression of five genes (TIL, ZIFL1, PGIP1, CSCL9 and MMI9.1) was increased by cold, but obviously delayed in comparison with that in A. thaliana (Fig. 8). Additionally, the transcriptional levels of six genes, including GRF2, RLP33, GORK, Q8W589, SERPIN1, and BGLU10, did not show any obvious change under cold treatment (Fig. 8). These results implied that the absence of CRT/DRE elements resulted in either non-response or postponed response in the expression of cold induced genes, resulting in the weak cold tolerance in C. equisetifolia. We also determined the expression of the CBF downstream genes containing DRE elements in C. equisetifolia, including ERD7, RD26, EXL2, HVA22D, LEA14, and RAV1. These genes exhibited induced but delayed expression under cold stress (Supplemental Fig. S4), indicating that CRT/DRE elements were contributed to their expression mediated by CBF.

    Figure 8.  The expression of downstream genes without DRE elements but directly regulated by CBF in C. equisetifolia, containing two similar expression pattern genes (GOLS1, NRT1.7), five delayed genes (ZIFL1, TIL, PGIP1, CSCL9, MMI9.1) and six weakly responsive and non-responsive genes (GBF2, RLP33,GORK,Q8W589, SERPIN1, BGLU10), GOLS1, galactinol synthase 1; NRT1.7, NITRATE TRANSPORTER 1.7; ZIFL1, ZINC INDUCED FACILITATOR-LIKE 1; TIL, temperature-induced lipocalin; PGIP1, POLYGALACTURONASE INHIBITING PROTEIN 1; GBF2, G-box binding factor 2; RLP33, receptor like protein 33; GORK, GATED OUTWARDLY-RECTIFYING K+ CHANNEL; SERPIN1, Inhibitor of pro-apoptotic proteases, which is involved in the regulation of the programmed cell death induction; BGLU10, BETA GLUCOSIDASE 10. The values are the mean ± standard deviation of three biological replicates. Relative expression in untreated plants (0 h) was set to 1. *p < 0.05, **p < 0.01.
    DownLoad: Full-Size Img PowerPoint

    The expression analysis of CBF upstream genes

    It has been reported that cold stress rapidly up-regulated the transcriptional expression of the CBFs through a complex signal transduction network in A. thaliana, involving transcription factor ICE1, CAMTAs, MYB15, Circadian Clock‐related components CCA1/LHY, and the light signaling components PIF3/4/7[6,62]. Based on transcriptome analysis, we discovered a stark contrast in the expression patterns of CBF genes between A. thaliana and C. equisetifolia under cold treatment. In A. thaliana, the expressions of three CBF genes were significantly upregulated at 2 h after the start of cold treatment. However, in C. equisetifolia, the noticeable upregulation of their homologous genes was delayed and occurred after 24 h of cold treatment (Fig. 9a). Besides that, few upstream genes of CBFs were identified and their expression profiles were compared to that of A. thaliana. These upstream genes could be divided into three types according to their expression pattern: the first group including genes non-induced by cold stress (PIF4, PIF7, CAMTA5, OST1), the second group including delayed induced genes (PUB25, MYB15, PRR5, JAZ1, LUX, EGR2, ELF4, COR28, COR27) while the third group including genes with similar expression patterns to those in A. thaliana (CCA1, TOC1) (Fig. 9a). Further, the transcription levels of these genes and three CeqCBFs were detected under chilling treatment at different time points using qPCR. The results displayed that compared to the expression of genes in A. thaliana, the obvious increase in the expression of CeqCBFs (CeqCBF1, CeqCBF2, CeqCBF3, CeqCBF4, 2 h vs 12 h), LUX (24 h vs 72 h), ELF4 (24 h vs 168 h), MYB15 (2 h vs 72 h), PRR5 (24 h vs 72 h), TOC1 (24 h vs 168 h) was obviously delayed (Figs 5b, 9b). Chilling treatment did not induce the expression of JAZ1, PIF4, PIF7, CCA1, and OST1. These results demonstrated that one of the factors for the cold stress sensitivity of C. equisetifolia could be delayed response or non-response of the upstream regulatory genes.

    Figure 9.  Comparison of upstream regulatory genes for CBF in A. thaliana and C. equisetifolia. (a) Heatmap of CBF upstream regulatory genes in A. thaliana and C. equisetifolia. (b) The expression of CBF upstream gene in C. equisetifolia. CBF, C-repeat/DRE binding factor; LUX, Homeodomain-like superfamily protein; ELF4, early flowering-like protein; MYB15, myb domain protein 15; PRR5, two-component response regulator-like protein; TOC1, CCT motif -containing response regulator protein; JAZ1, jasmonate-zim-domain protein 1; PIF4/7, phytochrome-interacting factor4/7; CCA1, circadian clock associated 1; OST1, Protein kinase superfamily protein. Relative expression in untreated plants (0 h) was set to 1. *p < 0.05, **p < 0.01.
    DownLoad: Full-Size Img PowerPoint

    Analysis on the cold intolerant mechanism in tropical and subtropical plants

    C. equisetifolia is one of the typical tropical and subtropical plants, exhibiting sensitivity to cold stress, which is also the property of most of the tropical and subtropical plants. According to the results of our analysis, the absence of CBFs binding motif located in the promoters of their downstream genes maybe one of the main reasons for C. equisetifolia' cold intolerance (Fig. 7). To investigate whether the loss of CRT/DRE elements is common to tropical and subtropical plants and how it affects the cold response, the homologous proteins of CBFs in different tropical and subtropical plants, including Theobroma cacao, Citrus sinensis, Citrus clementina, and Musa nana Lour, Citrus clementina, Citrus sinensis, Populus trichocarpa, and Hevea brasiliensis, were identified by MEGA11 and phylogenetic tree was constructed. The result of CBF conserved domains analysis showed that typical domains of CBFs in these homologous proteins were highly conserved (Supplemental Fig. S5), indicating that CBFs may play similar roles in response to cold stress in different tropical and subtropical plants. Further, CRT/DRE motif was analyzed in CBF's downstream target genes by using FIMO[49]. The number of CRT/DRE motif was significantly reduced in the detected promoter range of downstream genes in different species (Fig. 10), demonstrating that absence of CRT/DRE may be a common feature for tropical and subtropical plants.

    Figure 10.  CRT/DRE element analysis of downstream cold response genes directly regulated by CBF in other tropical and subtropical plants.
    DownLoad: Full-Size Img PowerPoint

    Discussion

    Cold acclimation is a vital strategy for improving plant cold resistance. Many tropical/subtropical plants, such as Litchi chinensis[63], Ananas comosus[64], Citrus[65], Musa spp[66], and C. equisetifolia[67], can gradually distribute and survive in wider geographic and temperature ranges through cold acclimation. In this study, whether acclimated or not, C. equisetifolia maintained a low relative ion leakage rate when exposed to −2 °C, −4 °C, and −6 °C for 2 h, respectively (Supplemental Table S3), demonstrating that C. equisetifolia had a certain degree of tolerance to freezing stress for short treatment time. However, with longer exposure to low temperature, the ion leakage rate dropped to 17.16% after treatment at −6 °C for 6 h, compared to 53.89% of non-acclimated C. equisetifolia seedlings. On the contrary, it was prominent difference in the survival rate between the NA plants (21.15%) and CA plants (59.02%) (Supplemental Table S3). Further, when treating plants with the following condition (at 4 °C for 4 d and then −8 °C for 6 h), the rate of survival and the ion leakage of C. equisetifolia seedlings was separately 5.22% and 45.84% (Supplemental Table S3, Fig. 1c, d). In contrast, under the same treatment condition, the survival rate of A. thaliana increased from 0 to 100% after cold acclimation (Fig. 1c). This proposed that cold acclimation can improve the cold resistance of C. equisetifolia to some extent although this improvement was significantly lower than that of A. thaliana. It is speculated that C. equisetifolia may lack certain low temperature response or regulatory factors that are present in A. thaliana.

    Comparing the low temperature transcriptomes of C. equisetifolia and A. thaliana showed that many low temperature responsive genes in C. equisetifolia exhibited no expression or weak or delayed response under low temperature stress, particularly in the CBF regulation pathway (Figs 4, 7 & 9). Further analysis revealed that numerous downstream target genes of CBF lacked the DRE binding element (Figs 7, 10), leading to influence the binding of CBFs to the downstream genes and then effect the expression of downstream genes, therefore decrease the cold adaptability of tropical and subtropical plants.

    In A. thaliana, cold acclimation triggers altering the expression of a large number of cold regulated genes[62]. According to whether directly regulated by CBF or not, these COR genes are divided into CBF-dependent pathway and CBF-independent pathway. Many studies have reported that different transcription factors and proteins are involved in CBF-dependent signal pathways[6]. It has been shown that plenty of COR genes, including KIN1/2, COR15A/B, LTI78[68,69], play major roles in regulating plant' cold response. Overexpressing of COR15A, encoding a chloroplast-targeted polypeptide in A. thaliana, results in a marked increase in the survival rate of separated protoplast frozen at −4.5 °C to −7 °C[70,71]. By comparing the homologous COR genes in C. equisetifolia and A. thaliana, we found that C. equisetifolia lacked homologous genes of COR15A, COR15B, HTT5, and STMP2 (Fig. 4), and the similar case was also discovered in other tropical/subtropical plants, such as Theobroma cacao, Musa nana Lour, Citrus clementina, Citrus sinensis, and Hevea brasiliensis (Fig. 11), speculating that the loss of key COR genes may be a crucial factor for their low temperature sensitivity and this may happen during the evolution process of tropical/subtropical plants in high temperature environments.

    Figure 11.  Homologous cold response genes in A. thaliana and other tropical/subtropical plants.
    DownLoad: Full-Size Img PowerPoint

    Cold-induced transcription factors CBF/DREB1 from AP2/ERF gene family respond to low temperature condition by directly modulating the expression of COR genes[47,59]. In Arabidopsis, the expression of CBFs shows rapid and early response to cold acclimation, and reaches the highest transcription level at 4 °C within 2 h[61,72]. Some studies have displayed that the expression of CBFs often exhibits a delayed response in Hevea brasiliensis, which is one of the typical tropical trees and sensitive to low temperature[32,34,73]. C. equisetifolia contained five CBF homologous genes, and cold acclimation highly induced the expression of CeqCBFs, which was accordance with the expression pattern of CBFs in A. thaliana, except that the peak expression of CeqCBFs occurred at 12 h after the start of cold acclimation (Fig. 5, Fig. 9b). In addition, CBF in C. equisetifolia, as well as in other tropical/subtropical plants, shared high conservation with Arabidopsis CBF in conserved structure domains, despite of certain differences in individual amino acid sites (Fig. 6a, Supplemental Fig. S5), suggesting that CBF maintained the conservation during the evolutionary process in plants at different latitudes. Nevertheless, our experiments had revealed that C. equisetifolia CBF could effectively restore the sensitive phenotype of Arabidopsis cbf1/2/3 triple mutants to low temperature conditions (Fig. 6d, Supplemental Fig. S3a), indicating that CBFs were functionally conserved in C. equisetifolia. Considering that cold acclimation may take a relatively long time, the postpone response of CeqCBFs may not be the primary factor causing its sensitivity to low temperatures. Therefore, the main reason for the cold intolerance of C. equisetifolia could be the absence of the CBF binding element (DRE) of the COR genes in C. equisetifolia and multiple tropical/subtropical plants (Fig. 7, Fig. 11). We speculated that long-term exposure to high-temperature environments may result in the loss of these elements during the evolutionary process of tropical/subtropical plants, thereby, leading to the sensitivity to low temperatures. It is worth noting that, both in C. equisetifolia and A. thaliana, COR genes-mediated by the low temperature at different time points exhibited a typical time-dependent cascade amplification. In detail, most early cold responsive genes quickly returned to the expression levels before induction, while other cold responsive genes started to respond (Fig. 2c). In such a situation, the delayed expression of CBF may affect this cascade regulation mode, and may also be one of the reasons for C. equisetifolia's cold intolerance.

    Previous studies focused on the certification of low temperature responsive genes in order to understand the molecular regulation mechanisms and to enhance plant cold resistance through overexpression these genes. By comparing and analyzing differences in low-temperature response between the model plant A. thaliana and C. equisetifolia, we hypothesized that the loss of DRE elements in multiple COR genes in C. equisetifolia was associated with its response to low temperatures, and the absence of key COR genes was also a significant factor contributing to C. equisetifolia's and other tropical/subtropical plants' sensitivity to low temperatures (Fig. 12). Therefore, introducing DRE elements into the related genes or expressing the absent genes in tropical/subtropical plants using modern biotechnological tools could be a new and important research ideas for improving the tropical/subtropical plants' cold resistance.

    Figure 12.  Comparative pattern plot of cold-response regulatory pathway in A. thaliana and C. equisetifolia.
    DownLoad: Full-Size Img PowerPoint

    Acknowledgments

    This work was supported by the National Key R&D Program for Young Scientists (grant number: 2021YFD2200900), the State Key Laboratory of Subtropical Silviculture (grant number: SKLSS-KF2022-08).

  • The authors declare that they have no conflict of interest.

  • [1]

    Liu H, Fan X, Yang L. 1998. Analysis of nutrient composition of fragrant fruit. Proceeding of The National Academic Conference on Natural Products and Flavors, 1998, Hainan. The Chinese Chemical Society, China. pp. 112−14
    [2]

    Wu Y, Li J, Tu P, Liu J, Chen J, et al. 2022. Introduction and cultivation techniques of persimmon in Guangdong. Guangdong Agricultural Sciences 49(7):42−48

    doi: 10.16768/j.issn.1004-874X.2022.07.006

    CrossRef   Google Scholar

    [3]

    Zhong Z, Yang J. 2008. Cultivation and management of white persimmon. Technical Services 19(3):4−7

    doi: 10.29571/ZHWHGX.200809.0002

    CrossRef   Google Scholar

    [4]

    Huang L. 2014. Main diseases and insect pests and their control measures. Southern Agriculture 8(30):32−35

    doi: 10.19415/j.cnki.1673-890x.2014.30.016

    CrossRef   Google Scholar

    [5]

    Zhang Y. 1986. Pollen longevity and storage. Plant Journal 1986(1):35

    Google Scholar

    [6]

    Su H, Li G. 2015. Effects of hand pollination on yield and quality of Pitaya fruit with red skin and meat. Jilin Agriculture 2015(7):77

    doi: 10.14025/j.cnki.jlny.2015.07.029

    CrossRef   Google Scholar

    [7]

    Li A, Li Z, Yuan B, Li Z, Zhou F. 2010. Effect of castration on maize yield traits. Journal of Shanxi Agricultural Sciences 2010(9):17−19

    doi: 10.3969/j.issn.1002-2481.2010.09.05

    CrossRef   Google Scholar

    [8]

    Guo P. 2019. How to configure the apple pollination tree. Shanxi Fruit Trees 2019(3):91

    doi: 10.3969/j.issn.1005-345X.2019.03.034

    CrossRef   Google Scholar

    [9]

    Zhou Z, Chen J, He J, Yu X. 2010. Development and application of plant in situ hybridization. Crop Research 2001(S1):50−55

    Google Scholar

    [10]

    Park H, Park SU, Jang B, Chung YS. 2020. Germplasm evaluation of Kenaf (Hibiscus cannabinus) for alternative biomass for cellulosic ethanol production. GCB Bioenergy 13:201−210

    doi: 10.1111/gcbb.12758

    CrossRef   Google Scholar

    [11]

    Khadka K, Raizada MN, Navabi A. 2020. Recent progress in germplasm evaluation and gene mapping to enable breeding of drought-tolerant wheat. Frontiers in Plant Science 11:1149

    doi: 10.3389/fpls.2020.01149

    CrossRef   Google Scholar

    [12]

    Narayanapur VB, Suma B, Minimol JS. 2018. Self-incompatibility: A pollination control mechanism in plants. International Journal of Plant Sciences 13:201−12

    doi: 10.15740/HAS/IJPS/13.1/201-212

    CrossRef   Google Scholar

    [13]

    Yabe S, Ohsawa R, Iwata H. 2014. Genomic selection for the traits expressed after pollination in allogamous plants. Crop Science 54:1448−57

    doi: 10.2135/cropsci2013.05.0319

    CrossRef   Google Scholar

    [14]

    Peter CI, Shuttleworth A. 2014. Catching on to concatenation: evidence for pre-pollination intra-sexual selection in plants. New Phytologist 203:4−6

    doi: 10.1111/nph.12838

    CrossRef   Google Scholar

    [15]

    Liede S, Hammer S, Whitehead V. 1991. Observations on pollination and hybridization in the genus Conophytum (Mesembryanthemaceae). Bradleya 9:93−99

    doi: 10.25223/brad.n9.1991.a3

    CrossRef   Google Scholar

    [16]

    Metelmann S, Sakai S, Kondoh M, Telschow A. 2020. Evolutionary stability of plant-pollinator networks: efficient communities and a pollination dilemma. Ecology Letters 23:1747−55

    doi: 10.1111/ele.13588

    CrossRef   Google Scholar

    [17]

    Hu G, Gao C, Fan X, Gong W, Hu F, et al. 2020. Pollination compatibility and xenia in Camellia oleifera. HortScience 55:898−905

    doi: 10.21273/HORTSCI14933-20

    CrossRef   Google Scholar

    [18]

    Lodolini EM, Polverigiani S, Bignozzi P, Santinelli A, Neri D, et al. 2018. Inflorescence characteristics and pollination compatibility of two local olive cultivars in high-density plantation. Acta Horticulturae 1229:243−48

    doi: 10.17660/actahortic.2018.1229.37

    CrossRef   Google Scholar

    [19]

    Lai H, Chen X, Chen Z, Zhou Y, Ou W, et al. 2014. In Situ pollen germination and artificial pollination compatibility in cassava (Manihot esculenta Crantz). Applied Mechanics and Materials 2512:245−51

    doi: 10.4028/www.scientific.net/amm.651-653.245

    CrossRef   Google Scholar

    [20]

    McLaren GF, Fraser JA. 1996. Pollination compatibility of ‘Sundrop’ apricot and its progeny in the 'Clutha' series. New Zealand Journal of Crop and Horticultural Science 24:47−53

    doi: 10.1080/01140671.1996.9513934

    CrossRef   Google Scholar

    [21]

    McLaren GF, Fraser JA, Grant JE. 1995. Pollination compatibility of apricots grown in central Otago, New Zealand. Acta Horticulturae 384:385−90

    doi: 10.17660/actahortic.1995.384.61

    CrossRef   Google Scholar

    [22]

    Zhang Y, Zhu J, Dai H. 2013. Morphological characteristics and pollination compatibility of a new red flesh apple, Hongxun No. 1. Research on Crops 14:199−204

    Google Scholar

    [23]

    Shivakumar M, Gireesh C, Talukdar A. 2016. Efficiency and utility of pollination without emasculation (PWE) method in intra-and inter-specific hybridization in soybean. Indian Journal of Genetics and Plant Breeding 76:98−100

    doi: 10.5958/0975-6906.2016.00013.4

    CrossRef   Google Scholar

    [24]

    Pandey AK, Pal AK, Shukla PK, Yadav MP. 2013. Germplasm evaluation of wood apple (Feronia limonia L.). Progressive Horticulture 45:76−79

    Google Scholar

    [25]

    Ashok K, Bist LD, Avasthe RK. 2010. Germplasm evaluation of low-chill pears (Pyrus spp.) for vegetative, reproductive and fruit quality parameters in Tarai area of Uttarakhand. Indian Journal of Plant Genetic Resources 23:310−14

    Google Scholar

    [26]

    Shweta, Ranjeet, Prakash. 2010. Assessment of germplasm resources in Indian Mustard. Asian Journal of Bio Science 5:140−41

    Google Scholar

    [27]

    Dennis FG Jr. 1997. Fruit cultivar and germplasm evaluation. HortScience 32:1007−10

    doi: 10.21273/HORTSCI.32.6.1007

    CrossRef   Google Scholar

    [28]

    Li X, Xu H, Feng J, Zhou X. 2015. Mining of genic SNPs and diversity evaluation of landraces in loquat. Scientia Horticulturae 195:82−88

    doi: 10.1016/j.scienta.2015.08.040

    CrossRef   Google Scholar

    [29]

    Yousef EAA, Lampei C, Schmid KJ. 2015. Evaluation of cauliflower genebank accessions under organic and conventional cultivation in Southern Germany. Euphytica 201:389−400

    doi: 10.1007/s10681-014-1225-y

    CrossRef   Google Scholar

    [30]

    Kumar B, Shukla A, Singh Y. 2013. Evaluation and characterization of germplasm accessions of urdbean (Vigna mungo L. Hepper). Trends in Biosciences 6(6):858−60

    Google Scholar

    [31]

    Mohammadi B, Khodadadi M, Karami E, Shaaf S. 2014. Variation in agro-morphological characters in Iranian garlic landraces. International Journal of Vegetable Science 20:202−15

    doi: 10.1080/19315260.2013.788594

    CrossRef   Google Scholar

    [32]

    Laserna-Ruiz I, De-Los-Mozos-Pascual M, Santana-Méridas O, Sánchez-Vioque R, Rodríguez-Conde MF. 2012. Screening and selection of lentil (Lens Miller) germplasm resistant to seed bruchids (Bruchus spp). Euphytica 188:153−62

    doi: 10.1007/s10681-012-0752-7

    CrossRef   Google Scholar

    [33]

    Stommel JR, Whitaker BD. 2003. Phenolic acid content and composition of eggplant fruit in a germplasm core subset. Journal of the American Society for Horticultural Science 128:704−10

    doi: 10.21273/JASHS.128.5.0704

    CrossRef   Google Scholar

    [34]

    Arzani K. 2021. The National Asian Pear (Pyrus serotina Rehd) project in Iran: Compatibility and commercial studies of introduced cultivars. Acta Horticulturae 1315:91−97

    doi: 10.17660/actahortic.2021.1315.14

    CrossRef   Google Scholar

    [35]

    Flores F, Gutierrez JC, Lopez J, Moreno MT, Cubero JI. 1997. Multivariate analysis approach to evaluate a germplasm collection of Hedysarum coronarium L. Genetic Resources and Crop Evolution 44:545−555

    doi: 10.1023/A:1008682019883

    CrossRef   Google Scholar

    [36]

    Kamal KS, Saleem M, Pardeep S. 2009. Variability studies on fruit characteristics of Terminalia bellirica. Journal of Research, SKUAST-J 8:86−91

    Google Scholar

  • Cite this article

    Lv W, Johnson JB, Zaman QU, Zhu M, Liu H, et al. 2023. Artificial pollination can improve fruit set and quality in the ice cream tree (Casimiroa edulis). Tropical Plants 2:12 doi: 10.48130/TP-2023-0012
    Lv W, Johnson JB, Zaman QU, Zhu M, Liu H, et al. 2023. Artificial pollination can improve fruit set and quality in the ice cream tree (Casimiroa edulis). Tropical Plants 2:12 doi: 10.48130/TP-2023-0012
    shu

Figures(5)  /  Tables(10)

Download PDF

Article Metrics

Article views(3429) PDF downloads(491)

Other Articles By Authors

ARTICLE   Open Access    

Artificial pollination can improve fruit set and quality in the ice cream tree (Casimiroa edulis)

  • 1.

    Sanya Nanfan Research Institute of Hainan University, Hainan Yazhou Bay Seed Laboratory, Sanya 572025, China

  • 2.

    Collaborative Innovation Center of Nanfan and High-Efficiency Tropical Agriculture, School of Tropical Crops, Hainan University, Haikou 570228, China

  • 3.

    School of Health, Medical & Applied Sciences, CQ University Australia, Bruce Hwy, North Rockhampton, QLD 4701, Australia

  • 4.

    Hainan Shengda Modern Agriculture Development Co., Ltd., Qionghai 571425, China

  • Received: 19 September 2022
  • Accepted: 19 July 2023
  • Published online: 10 August 2023
Tropical Plants  2,  Article number: 12  (2023)  |  Cite this article

Abstract: Ice cream fruit (Casimiroa edulis La Llave) is a member of the Rutaceae family native to South America, which has recently been introduced to China. In order to improve the yield of ice cream fruit, we carried out a hybridization experiment with paired pollination. Pollen was collected in the morning and carefully stored, before it was applied to the stigmata of sprouted flowers, using a cotton swab or tweezers. This artificial pollination is repeated at different times of the day. This was continued until the stamens fall off the flowers, the stigmata withered, and the fruit began to develop. Fruit yield and quality were recorded as the principal metrics of the success of each hybrid. At the Hainan Tropical Fruit Window Agricultural Company, nine different ice cream fruit varieties were selected and divided into three groups (abundant pollen, little pollen and no pollen) according to the amount of pollen that each variety naturally produced. Four experimental conditions were investigated: 1. No artificial pollination (natural self-pollination); 2. Artificial self-pollination; 3. Cross-pollination (no emasculation); 4. Cross-pollination (with emasculation). The results indicated that the fruit from cultivars producing abundant pollen generally had poor quality, smaller size, average sweetness, and a medium rate of natural fruit set. In contrast, cultivars producing little pollen showed better fruit quality, higher fruit fertility and increased success rates for artificial pollination. The N2 ice cream fruit cultivar showed the best characteristics as the paternal line (pollen source), while the 4P cultivar showed the best characteristics as the maternal line (flower source). The New varieties (N2 × 4P) performs well in fruit quality, fruit size, fruit sweetness and fruit setting rate. Consequently, this cross-pollinated cultivar is recommended for agricultural production and planting.

    HTML

    Introduction

      Origin and development of ice cream fruit

    • Ice cream fruit (Casimiroa edulis La Llave) is a tropical fruit tree from the Rutaceae family, also known by the common names white persimmon, white sapote, Mexican apple and fragrant meat fruit. It is a monoecious species, bearing male and female parts in the same flowers (existing simultaneously)[1]. It is called ice cream fruit in China because it tastes like vanilla ice cream after freezing. Ice cream fruit originated in Mexico and Central America, but is currently distributed across a range of tropical and subtropical regions[2]. The tree shows high levels of resistance against biotic and abiotic stresses[3]. The fruit is rich in flavor, with soft flesh, minimal fruit juice, a milk-like fragrance, and a sweet flavor reminiscent of ice cream[1]. This combination of characteristics makes them highly sought after by consumers.

      At present, the commercial planting area of domestic ice cream fruit is not large, and the output is limited, resulting in a relatively high market price of around 50 Euros/kilogram (390 Chinese Yuan/kg). Similarly, it is not widely planted in China, only being introduced in Hainan, Yunnan and a few other places. However, as a tropical fruit with high economic value, there is considerable promise to expand its production as a high-end tropical fruit variety in the domestic market. Therefore, it is necessary to further study the cultivation technology of ice cream fruit - cross pollination.

      Morphology and floral biological characteristics of ice cream fruit

      Morphological characteristics

    • In the Hainan region, ice cream fruit trees can grow to 5−6 m, with gray-brown bark. The leaves are oval and lanceolate when young, with 3−5 leaflets typically comprising each leaf. They range between 6–12 cm long and 4–6 cm wide, with short-pointed or rounded leaf tips. The leaf margin has fine blunt teeth, with short hairs on the petiole, leaf shaft. The fruit begins green, turning more spherical and yellow and spherical as it ripens[3].

      The flowers have five petals, with a yellow-green corolla borne at the leaf base, around 8−10 mm in size. The stamens are 2−3 mm long.

      The medium-sized (5−10 cm diameter) fruit is shaped like a persimmon, with thin skin, white or yellowish flesh, and a smooth texture (Fig. 1). When refrigerated, it tastes like ice cream, with a sweetness of 17%−25%. A mature tree can produce 500 kg of fruit in one fruit period.

      Figure 1. 

      (a) Ice cream fruit. (b) Peeled ice cream fruit. (c) Ice cream tree flower.

      It is generally easy to cultivate, growing in a range of conditions (light, shaded, dry, wet, barren soil). It shows good cold resistance, withstanding temperatures as low as −2 to −4 °C.

      Floral characteristics

    • Ice cream fruit is widely cultivated in the tropics and subtropics, and can also flower in temperate regions. Ice cream fruit can bear fruit more than once per year, with flowering occurring twice a year between January to February, and June to July[2]. The time from flower germination until flowering is around 15−20 d[1], but fertilisation rates and fruit survival vary widely among cultivars. Varieties producing little pollen or no pollen seem to have a lower fruit yield but a higher fruit survival rate; Varieties with more pollen have lower fruit survival rate.

      Ice cream fruit nutritional composition

    • Ice cream fruit contains a lot of trace elements beneficial to the human body, such as potassium, calcium, iron and other minerals. It also contains high levels of several vitamins, including vitamin C (62 mg vitamin C/100 g); in some areas this fruit is colloquially known as the 'king of vitamin C'. Ice cream fruit is high in sugar, including fructose, glucose and other sugars, with a total sugar content as high as 16%[4].

      Research status of ice cream fruit

    • Practical research into any crop is inseparable from the collection of germplasm resources, breeding techniques and the investigation of biotic and abiotic factors affecting it. At present, few research groups are conducting experiments on ice cream fruit in China, thus much basic experimental research and investigation is still required.

      In China, ice cream fruit is currently cultivated on a large scale in Yunnan Xishuangbanna Botanical Garden and in the Hainan Shanda agricultural tropical fruit window. This survey uses ice-cream fruit grown by Hainan Qionghai Shanda agricultural company. The imported ice cream fruit came from the United States. This is one of the few only plantings areas in China.

      Study purpose and significance

    • At the present time, commercial ice cream fruit plantings suffer from low to moderate fruit set rates following artificial pollination, as well as inconsistencies in fruit quality. In order to improve the yield and quality of ice cream fruit, we investigated different artificial pollination methods in this study. Different ice cream fruit varieties were crossed using the artificial hybrid method for paired pollination experiments, with pollen taken from one variety used to pollinate another variety. The resultant fruit yield and quality were used as metrics to determine the best-performing crosses.

      Main content of the study

    • Ice cream fruit is a monoecious species, and pollen counts vary depending on the variety. A total of nine varieties were investigated in this work, being divided into three groups: little pollen (varieties AU4, 4P, C1), abundant pollen (N2, AU1, AU2) and no pollen (N6, N7, W4). The varieties with abundant pollen or little pollen were used as male parents, while the varieties with little pollen or no pollen were used as the female parents. In addition to cross-pollination, natural hanging fruit and artificial self-pollination were also investigated in each variety. After pollination, the flowers were bagged to avoid interference from external factors.

    Results

      Observation of natural self-pollination and artificial self-pollination

    • The different ice cream fruit varieties showed significant morphological differences in their stamens, as shown in Fig. 2.

      Figure 2. 

      (a) W4 variety – no pollen. (b) 4P variety – little pollen. (c) AU2 variety – abundant pollen.

      The varieties with no pollen showed large ovaries, with a visible, yellow pistil stigma. The stamens and yellow and black, with dry and infertile anthers.

      The varieties with little pollen stigma is pink or yellow-pink, and the ovary and stigma are abnormally large. Five stamens are very small, the anthers are yellow-white or yellow-black, and the pollen content is very little.

      The varieties with abundant pollen pistil is very small, and the stigma is not obviously light green, the capital part is also very small, but the five stamens are very thick, especially the anther flower sac is also very large, showing bright yellow, the pollen content is very high.

      In Fig. 3, the development process of naturally self-pollinated fruit is recorded, with the corresponding data for this experimental group shown in Table 1.

      Figure 3. 

      The natural development process of self-pollinated fruit.

      Table 1.  Statistics of the naturally self-pollinated fruit.

      BreedRate of fruit set
      (%)      		Width
      (cm)      		Length
      (cm)      		Sugar
      (%)
      Abundant pollenAU10%−−−
      AU225%4.1 ± 04 ± 010.7 ± 0
      N250%4.7 ± 0.23.95 ± 0.259.1 ± 0.1
      Little pollen4P66%7.5 ± 0.26.9 ± 0.111.7 ± 0.5
      AU450%8.3 ± 0.27.6 ± 0.310.5 ± 05
      C1100%6.6 ± 0.25 ± 0.110.9 ± 0.1

      Comparing the two groups (abundant pollen and little pollen) shows that the fruit set rate, fruit size and sweetness were all higher in varieties with little pollen, compared to those with more abundant pollen. Within each group, variety N2 showed the best performance for the abundant pollen varieties, while variety 4 showed the highest sweetness and acceptable size and fruit set amongst the varieties with little pollen. However, variety C1 did show the highest rate of fruit set.

      To compare the effects of natural and artificial pollination, another experiment was carried out with artificial self-pollination conducted at different times every day. The bag surrounding each flower was gently shaken each time to ensure that the pollen was transferred from the stamens to the stigma; this process was repeated until the stamens and stigma withered, and the fruit began to grow, indicating that pollination was successfully completed.

      As shown in Table 2, the fruit set rate of ice cream fruit varieties with abundant pollen increased in the case of artificial pollination. However, there was no significant change in the size or sweetness of the fruit quality. It is known that artificial pollination can slightly improve the fruit set rate of abundant pollen varieties, but it has no significant effect on fruit quality.

      Table 2.  Statistics of experimental data of artificial self pollination.

      BreedFruit set
      (%)      		Width
      (cm)      		Length
      (cm)      		Sugar
      (%)
      Abundant pollenAU133%5.9 ± 05.5 ± 010.0 ± 0
      AU250%4.4 ± 0.54.0 ± 0.19.5 ± 0.5
      N240%6.0 ± 04.5 ± 08.6 ± 0
      Little pollen4P66%7.7 ± 0.16.4 ± 0.811.0 ± 1.0
      AU450%8.9 ± 0.17.5 ± 0.110.2 ± 0.2
      C1100%7.8 ± 0.16.9 ± 0.19.5 ± 0.1

      Therefore, artificial cross-pollination and hybridization studies were conducted to find possible combinations which could provide improved fruit quality compared to self-pollination.

      Artificial cross-pollination (male/female)

    • Tables 3−8 show the experimental results from the artificial cross-pollinations, broken down by each female parent. Additionally, each table shows the results with and without emasculation.

      Table 3.  Experimental data for the cross-pollination with AU4 as the maternal parent, with and without emasculation.

      GroupMother treeFather treeFruit set (%)Length (cm)Width (cm)Sugar (%)
      FemaleAU4 (little pollen)Little pollenC133%7.5 ± 0.17.4 ± 0.18.9 ± 0.1
      4P50%8.8 ± 0.27.9 ± 0.210.4 ± 0.1
      Abundant pollenAU140%8.15 ± 0.17.75 ± 0.110.4 ± 0.2
      AU250%9.2 ± 0.17.95 ± 0.19.5 ± 0.1
      N275%9.47 ± 0.18.47 ± 0.110.5 ± 0.5
      MaleLittle pollenC10%−−−
      4P50%8.05 ± 0.17.95 ± 0.28.5 ± 1
      Abundant pollenAU133%8.7 ± 0.28.5 ± 0.18.0 ± 2
      AU233%8.5 ± 0.37.9 ± 0.18.1 ± 0.2
      N250%9.2 ± 0.38.04 ± 18.5 ± 1

      Table 4.  Experimental data for the cross-pollination with 4P as the maternal parent, with and without emasculation.

      GroupMother treeFather treeFruit set (%)Length (cm)Width (cm)Sugar (%)
      Female4P (little pollen)Little pollenC150%7.8 ± 0.67.0 ± 0.29.8 ± 1.2
      AU466%6.9 ± 0.16.3 ± 0.28.78 ± 1.0
      Abundant pollenAU150%8.7 ± 0.66.57 ± 1.09.8 ± 0.5
      AU250%7.35 ± 0.26.45 ± 2.08.5 ± 1.5
      N280%7.7 ± 1.07.075 ± 1.011.0 ± 1.0
      MaleLittle pollenC142%8.5 ± 0.36.9 ± 0.210.5 ± 0.5
      AU450%8.0 ± 2.07.95 ± 2.010.0 ± 0.8
      Abundant pollenAU150%7.15 ± 2.06.55 ± 3.09.2 ± 0.6
      AU242%6.8 ± 0.46.1 ± 0.58.87 ± 0.4
      N262%7.5 ± 0.37.4 ± 0.69.84 ± 0.3

      Table 5.  Experimental data for the cross-pollination with C1 as the maternal parent, with and without emasculation.

      GroupMother treeFather treeFruit set (%)Length (cm)Width (cm)Sugar (%)
      FemaleC1 (little pollen)Little pollenAU460%7.27 ± 1.06.76 ± 1.010.3 ± 1.0
      4P33%6.9 ± 0.45.9 ± 0.310.0 ± 0.5
      Abundant pollenAU125%6.8 ± 0.56.2 ± 0.68.0 ± 0.4
      AU233%5.9 ± 0.45.9 ± 0.310.2 ± 1.0
      N250%6.8 ± 0.66.8 ± 0.311.0 ± 0.5
      MaleLittle pollenAU450%6.9 ± 0.26.8 ± 0.410.3 ± 1.0
      4P0%−−−
      Abundant pollenAU10%−−−
      AU233%6.8 ± 0.45.9 ± 0.110.0 ± 0.5
      N250%7.1 ± 0.55.9 ± 0.29.16 ± 1.0

      Table 6.  Experimental data for the cross-pollination with N6 as the maternal parent, with and without emasculation.

      GroupMother treeFather treeFruit set (%)Length (cm)Width (cm)Sugar (%)
      FemaleN6 (No pollen)Little pollenC10%−−−
      4P40%7.0 ± 0.56.5 ± 0.28.75 ± 1.0
      AU450%6.8 ± 0.26.3 ± 0.210.0 ± 0.4
      Abundant pollenAU150%7.2 ± 0.25.9 ± 0.38.2 ± 0.2
      AU233%5.8 ± 0.46.2 ± 0.48.0 ± 1.0
      N250%6.9 ± 0.26.7 ± 0.310.0 ± 0.6
      MaleLittle pollenC150%6.6 ± 06.5 ± 08.0 ± 0
      4P33%7.4 ± 06.5 ± 09.5 ± 0
      AU433%6.5 ± 0.156.4 ± 0.48.0 ± 1.0
      Abundant pollenAU166%6.6 ± 0.55.95 ± 0.19.5 ± 0.5
      AU250%6.6 ± 0.16.45 ± 0.10.59 ± 0.5
      N266%6.6 ± 0.65.95 ± 0.110.5 ± 1

      Table 7.  Experimental data for the cross-pollination with N7 as the maternal parent, with and without emasculation.

      GroupMother treeFather treeFruit set (%)Length (cm)Width (cm)Sugar (%)
      FemaleN7 (No pollen)Little pollenC150%6.8 ± 0.26.4 ± 0.210 ± 0.4
      4P33%7.2 ± 0.26.8 ± 0.29.4 ± 0.4
      AU433%6.4 ± 0.26.2 ± 0.49.4 ± 0.4
      Abundant pollenAU133%6.7 ± 0.36.6 ± 0.610.0 ± 0.2
      AU233%7.8 ± 0.46.8 ± 0.59.0 ± 1.0
      N250%8.8 ± 0.47.4 ± 0.19.5 ± 1.0
      MaleLittle pollenC128%5.8 ± 0.15.6 ± 08.5 ± 0.5
      4P50%7.2 ± 06.8 ± 010.0 ± 0
      AU425%6.4 ± 06.3 ± 08.4 ± 0
      Abundant pollenAU10%−−−
      AU240%6.8 ± 06.1 ± 09.0 ± 0
      N250%6.8 ± 0.46.0 ± 0.210.0 ± 1.0

      Table 8.  Experimental data for the cross-pollination with W4 as the maternal parent, with and without emasculation.

      GroupMother treeFather treeFruit set (%)Length (cm)Width (cm)Sugar (%)
      FemaleW4 (No pollen)Little pollenC160%7.2 ± 06.5 ± 016.0 ± 0
      4P50%6.3 ± 0.35.8 ± 0.113.5 ± 0.5
      AU40%−−−
      Abundant pollenAU150%5.6 ± 05.4 ± 0.210.7 ± 0.3
      AU250%7.1 ± 06.2 ± 013.0 ± 0
      N260%6.8 ± 0.26.0 ± 0.114.2
      MaleLittle pollenC125%7.1 ± 0.36.4 ± 0.213.7 ± 1.0
      4P50%7.0 ± 0.16.4 ± 011.7 ± 0.6
      AU40%−−−
      Abundant pollenAU150%6.9 ± 06.8 ± 012.8 ± 0
      AU250%5.8 ± 04.9 ± 013.6 ± 0
      N260%7.1 ± 0.66.0 ± 0.312.8 ± 0.2

      When the AU4 variety was used as the maternal parent (with emasculation), the best performance was found using N2 as the pollen source, which yielded a 75% rate of fruit set and the largest fruit size (9.5 cm × 8.5 cm), as shown in Table 3. It also had a high sugar content (10.5%).

      Similarly, for 4P as the maternal parent, 80% fruit set was found with emasculation and with N2 as the pollen source (Table 4). This cross also showed moderate size and high sugar content. However, the largest fruit were found using AU1 as the pollen source.

      The final little-pollen variety used as a maternal parent was the C1 variety, which generally showed poorer performance than the other two maternal parents previously discussed.

      The best performance was seen using AU4 pollen on emasculated flowers, which gave a 60% rate of fruit set, the largest fruit size and a high sugar content (Table 5). N2 performed acceptably as a pollen source, and was also the best cross on non-emasculated flowers.

      For the maternal parent varieties which did not produce pollen, N7 generally had poorer performance, while N6 and W4 had acceptable performance. Again, N2 was the best pollen source for most maternal varieties.

      With N6 as the maternal parent, N2 and AU1 tied for the highest fruit set rate (66%) under non-emasculated conditions; however, the fruit was only moderate in size (Table 6). N2 also gave the sweetest fruit.

      For N7 as the maternal parent, several combinations gave a 50% fruit set rate, while most other pollen sources gave quite poor rates of fruit set (Table 7). The better-performing pollen sources included C1 and N2 (for emasculated flowers) and 4P and N2 (for non-emasculated flowers). N2 also gave the largest fruit under emasculated conditions (8.8 cm × 7.5 cm).

      Finally, with the W4 maternal parent, N2 again performed well under both emasculated and non-emasculated conditions (60% fruit set rate), with moderately sized fruit (Table 8). C1 also gave 60% fruit set when applied to emasculated flowers and good-sized fruit, as well as an exceptionally high sugar content (16%).

      In general, most crosses showed higher fruit set rates and better fruit quality when the stamen is emasculated. The index of fruit quality as the best paired pollination group is a general method for pollination variety research[5], so we calculated the formula of the best paired parent variety of each parent to obtain the index.

      Therefore, the best matching parent and the fruit quality of each parent in the case of male elimination can be shown as in Table 9. Overall, the best pollination combination between different ice cream fruit varieties was found using variety 4P and N2 as the parents (Table 9).

      Table 9.  Male parent cultivars and index of the best cultivar pairing for each female parent cultivar with emasculation.

      CrossFruit set (%)Length (cm)Width (cm)Sugar
      (%)      		Index of
      fruit quality
      Mother treeFather tree
      C1AU460%7.2 ± 1.06.7 ± 1.010.3 ± 1.010.2
      4PN280%7.7 ± 1.07.1 ± 1.011.0 ± 1.014.4
      AU4N275%9.4 ± 0.18.4 ± 0.110.5 ± 0.514.2
      N6N250%6.6 ± 0.65.9 ± 0.110.5 ± 1.08.3
      N7N250%8.8 ± 0.47.4 ± 0.19.5 ± 1.08.5
      W4C160%7.2 ± 06.5 ± 016.0 ± 013.5

      Hybrid fruit of 4P and N2

    • For the best-performing artificially pollinated hybrid between varieties 4P and N2, the fruit weight was 280 g, with a longitudinal width of 8.1 cm and a transverse diameter of 8.5 cm. This combination showed a 88.9% fruit set rate, with a 62.5% fruit survival rate. The skin of the fruit was green, with golden-yellow flesh, and a sugar content of 12%. This new variety has obvious advantages in both fruit quality and yield, and it shows great potential in the future agricultural production process (Fig 4).

      Figure 4. 

      Hybrid fruit of 4P and N2.

    Discussion

      Ice cream fruit flower differences

    • To gain further insight in the reproductive biology of ice cream fruit, a number of comparative observations on the flower morphology and growth are reported here.

      Ice cream fruit varieties with abundant pollen showed a large number of flowers, although with very small ovaries and stigmas. However, the anthers were large. Each branch can bear up to 12−20 flowers.

      Varieties with little pollen had significantly reduced numbers of flowers, although the ovaries were larger. Each branch bore 8–12 flowers.

      Finally, the ice cream fruit varieties with no pollen had an unusually large ovary, around 2−4 times larger than that of the other varieties flowering at the same period. The anthers are infertile and do not produce pollen. The stigmata on the pistil is also very large, after successful pollination, the stigma withers gradually.

      Pollination of different varieties of ice cream fruit

    • In natural self-pollination experiment, the following conclusions could be drawn:

      (1) Ice cream fruit varieties with abundant pollen generally show lower fruit sizes, average to low sweetness, and low to moderate rates of fruit set.

      (2) The ice cream fruit of little pollen varieties has good fruit quality and the fruit set rate is relatively high.

      Furthermore, we observed that in the absence of artificial intervention, ice cream fruit setting rate, fruit quality and sweetness were worse. This indicates that the ice cream fruit benefits from manual pollination intervention.

      In the experiments with artificial cross-pollination, we observed an increase in fruit set rate but no change in fruit sweetness or quality. This indicates that the ice cream fruit may have the characteristics of self-incompatibility, which limits the development of ice cream fruit related industries. In order to increase the yield, we must cross-pollinate and cross to find the best pair of pollinators. It is suggested that mixed pollen should be used for artificial pollination when growing fruits in the future[6]. The experimental results are consistent with this view.

      The experimentation results from artificial cross-pollination (Tables 4−9) showed that the fruit set rate, fruit size and sweetness were all improved. Based on the cross-pollination results from different varieties, we can conclude:

      Firstly, the best-performing variety as the male parent (i.e., the pollen source) is the variety N2, one of the abundant pollen varieties. In general, varieties producing more high pollen showed better performance as the pollen source, and can be used as the pollen source tree in agricultural production.

      Second, the best-performing variety for the female parent was variety 4P, which was one of the varieties bearing little pollen. It showed high bud quality and good fruit characteristics, recommending its use as the mother fruit tree in agricultural production and planting.

      Third, the combination of the pollen-free variety W4 as the mother tree and the little-pollen variety C1 as the pollen source tree showed a higher sweetness in their hybrid fruits than that of any other hybrid. This combination could be used as a special, high-value hybrid to enrich the agricultural production of ice cream fruit.

      Finally, male group and female group contrast. The fruit set rate, fruit quality and fruit sweetness were better in the female group. In the process of agricultural production, artificial cross pollination needs to emasculation. Other research has shown that the average yield of the same maize variety was increased significantly by emasculation than by no emasculation[7]. The experimental results are consistent with this view.

      Selecting appropriate varieties for cross-pollination is one of the key technologies to obtain high quality and high yield[8]. Hybrid technology has played an important role in plant genetics and breeding[9]. In particular, the abundant pollen variety N2 significantly improved fruit quality when used as a pollen source in this study, particularly when combined with variety 4P as the mother fruit tree.

    Materials and methods

      Materials, main instruments and equipment

      Test materials

    • The experimental field work was conducted at the Tuhao field station and Houpo field station, both owned by Hainan Shengda Modern Agricultural Development Co, Ltd, (Hainan, China).

      The ice cream fruit varieties used in this experiment were three varieties producing abundant pollen (N2, AU1, AU2) and three varieties producing little pollen (AU4, 4P, C1) and three varieties producing no pollen (N6, N7, W4). The varieties used in this experiment were randomly selected in the experimental field. All the experimental varieties were planted at appropriate temperature, humidity, sufficient light, with optimum growth conditions and no signs of abnormal flowers and fruits[10,11]. The tree were kept free of diseases and insect outbreaks during the trial.

      Instrument, equipment and appliances

    • The handheld glucose meter I used was the Nohawk HP-TD1.

      Test method

      Observation of natural self-pollination

    • No artificial interventions were made for the natural self-pollination experiment[12,13]; only the selected ice cream fruit trees and the sample flowers in the flower germination period were marked. Observations were made throughout the natural flowering and fruit set process. The fruit set rate and fruit quality data were also collected. In this experimental group, nine flowers were documented from each variety, for three abundant pollen varieties (N2, AU1 and AU2), three little-pollen varieties (AU4, 4P and C1). This gave a total of six experimental groups and 54 data points.

      Artificial self-pollination and cross-pollination (male/female)

    • For the artificial self-pollination work, the selected ice cream fruit trees and the sample flowers were marked, bagged and isolated during the flower germination period. The flowers were then artificially self-pollinated (i.e., between the flowers of the same fruit tree). For this experiment, nine flowers were recorded from each variety, for the three abundant pollen varieties (N2, AU1 and AU2) and the three little-pollen varieties (AU4, 4P and C1). Again, this gave six experimental groups and 54 data points.

      For the artificial cross-pollination work, one fruit tree of either an abundant pollen variety (N2, AU1, AU2) or a little-pollen variety (AU4, 4P and C1) was selected as the 'father' to collect pollen from. The varieties producing no pollen (N6, N7, W4) or little pollen (AU4, 4P and C1) were selected as mother trees (i.e., the flower source).

      For each of the six female trees used as the mother, either five or six other varieties were used as the pollen source to cross-pollinate the parent. Five cross-pollinations were performed where the mother was a little-pollen variety, while six cross-pollinations were performed where the mother was a no-pollen variety. Consequently, there were a total of 15 crosses for the little-pollen mother varieties, and 18 crosses for the no-pollen mother varieties, as shown graphically in Fig. 5. Again, nine flowers were tracked for each cross-pollination experiment.

      Figure 5. 

      Artificial cross-pollination (male/female).

      As a further investigation, this experiment was divided into two groups: male and female. This gave a total of 66 experimental treatments, yielding data on 594 flowers and fruit (Fig. 5).

      Experimental operation procedures

      Selected varieties (fruit trees)

    • For the biological and morphological observations, a total of 24 fruit trees were observed for different purposes (Table 10). The nine ice cream fruit varieties are planted in a large area in Hainan Shengda Agricultural Company, which belongs to the current commercial varieties[14]. The paired pollination completion experiments and experimental results are of great significance to their commercial production[15,16].

      Table 10.  Fruit tree different applications.

      Different varietiesDifferent applications
      Abundant pollen varieties (N2, AU1 and AU2)Observation of natural self-pollination3
      Artificial self-pollination3
      Artificial cross-pollination parent tree3
      Little-pollen varieties (AU4,
      4P and C1)      		Artificial cross-pollination mother tree (male)3
      Artificial cross-pollination mother tree (female)3
      Artificial cross-pollination parent tree3
      No pollen varieties (N6, N7 and W4)Artificial cross-pollination mother tree (male)3
      Artificial cross-pollination mother tree (female)3
      Sum24
      Selection of the flowers

    • From each tree, three flowers were randomly selected and marked for observations[17]. The flowers were bagged in the artificial self-pollination and cross-pollination experiments to avoid external factors[18,19].

      Pollen collection and preservation

    • Ice cream fruit pollen from the required parent was collected at the morning of the crossing, to avoid little pollen quality and quantity[20,21]. Using tweezers, the pollen was gently collected into separate bags for preservation[22]. No pollen was collected on rainy days, as moisture can affect pollen quality[23].

      Artificial pollination

    • Artificial self-pollination was carried out using bagged flowers[24]. Pollen from the required variety was gently shaken onto the stigma; this was repeated at different times every day until the stamens withered or fell off[25−27].

      Artificial cross-pollination was also carried out on bagged flowers; however, the stamens were removed (where required) at the germination stage[28,29]. As with the artificial self-pollination work, either cotton swabs or tweezers were used to transfer pollen from the required variety to the stigma of the mother flower at different times each day. This was repeated until the stamens fell off and the stigmata withered[30−32].

      Emasculation

    • For experiments where emasculation was required, the stamens of the flower were gently cut off at the germination stage. Care was taken to ensure the rest of the pistil and stigma were not harmed during this process[33,34].

      Measurement statistics

    • When the fruit ripened, it was picked and the length and width measured. The sugar content was also measured[35]. Finally, the relevant data was transferred into Microsoft Excel for subsequent processing and analysis[36].

      Acknowledgments

      • This study was funded by Hainan Province Science and Technology Special Fund (ZDYF2022XDNY190), the Project of Sanya Yazhou Bay Science and Technology City (SCKJ-JYRC-2022-83) , University level scientific research project of Hainan University (XTCX2022NYB09), and Hainan Provincial Natural Science Foundation of China (421RC486).

      Conflict of interest

      • The authors declare that they have no conflict of interest.

      Dates

      • Received 19 September 2022; Accepted 19 July 2023; Published online 10 August 2023

      Rights and permissions
      • Copyright: © 2023 by the author(s). Published by Maximum Academic Press on behalf of Hainan University. This article is an open access article distributed under Creative Commons Attribution License (CC BY 4.0), visit https://creativecommons.org/licenses/by/4.0/.
    Figure (5)  Table (10) References (36)
  • About this article
    Cite this article
    Lv W, Johnson JB, Zaman QU, Zhu M, Liu H, et al. 2023. Artificial pollination can improve fruit set and quality in the ice cream tree (Casimiroa edulis). Tropical Plants 2:12 doi: 10.48130/TP-2023-0012
    Lv W, Johnson JB, Zaman QU, Zhu M, Liu H, et al. 2023. Artificial pollination can improve fruit set and quality in the ice cream tree (Casimiroa edulis). Tropical Plants 2:12 doi: 10.48130/TP-2023-0012
    shu

Catalog

  • HTML

  • Introduction

  • Origin and development of ice cream fruit

  • Morphology and floral biological characteristics of ice cream fruit

  • Morphological characteristics
  • Floral characteristics

  • Ice cream fruit nutritional composition

  • Research status of ice cream fruit

  • Study purpose and significance

  • Main content of the study

  • Results

  • Observation of natural self-pollination and artificial self-pollination

  • Artificial cross-pollination (male/female)

  • Hybrid fruit of 4P and N2

  • Discussion

  • Ice cream fruit flower differences

  • Pollination of different varieties of ice cream fruit

  • Materials and methods

  • Materials, main instruments and equipment

  • Test materials
  • Instrument, equipment and appliances

  • Test method

  • Observation of natural self-pollination
  • Artificial self-pollination and cross-pollination (male/female)

  • Experimental operation procedures

  • Selected varieties (fruit trees)
  • Selection of the flowers
  • Pollen collection and preservation
  • Artificial pollination
  • Emasculation
  • Measurement statistics
  • Acknowledgments
  • Conflict of interest
  • Dates
  • Rights and permissions
  • About this article
logo

Contents

Policies

Services

Partnerships

© 2025 Maximum Academic Press, except Open Access articles. MAP is a member of ALPSP, COPE, CrossRef, cOAlition S, OASPA and STM.

Privacy Policy  |  Site Map  |  Terms and Conditions

/

DownLoad:  Full-Size Img  PowerPoint
Return
Return