Special Article: Flower Development

Ann Agric Crop Sci. 2023; 8(2): 1130.

Determination of the Microspore Development Concerning Floral Morphology for Improving Drought Tolerant Citron Watermelon (Citrullus lanatus var. citroides) Rootstocks Via Androgenesis.

Kurtar ES¹,*, Seymen M¹; Alan AR², Toprak FC²; Atakul Z³; Metin D³; Lachin AR⁴; Yirmibes B⁴

¹Department of Agriculture Faculty and Horticulture, Selcuk University, Turkey

²Plant Genetics and Agriculture Biotechnology Application and Research Center, Pamukkale University, Turkey

³Department of Horticulture, Institute of Science, Selcuk University, Turkey

⁴Institute of Science, Pamukkale University, Turkey

*Corresponding author: Kurtar ES Selcuk University, Horticulture Department of Agriculture Faculty, Konya, Turkey. Email: [email protected]

Received: March 20, 2023 Accepted: April 26, 2023 Published: May 03, 2023

Abstract

The negative effects of abiotic stress factors (especially drought and salinity) that arise due to global climate change are increasing in today’s agricultural production. To eliminate the negative effects of these stress factors, some cultural practices have been implemented such as the breeding of tolerant cultivars and rootstock, exogenous applications of PGPR, AMF, and chemical substances, etc. Contrary to exogenous applications, providing tolerance with breeding methods has an important role in efficient and sustainable agriculture. Androgenesis is a phenomenon for the induction and development of microspore-derived pure lines, as initial material for F1 cultivar breeding, in different vegetable crops. Success in microspore embryogenesis is considerably related to the culture of microspores at an appropriate developmental stage. There is no standard protocol for the anther culture in citron watermelon (Citrullus lanatus var. citroides). Thus, a relation between floral morphology (bud length and width, bud index, sepal position, petal color), and microspore development (microspore diameter, uninucleated and binucleated phases) was investigated in citron. The male flower buds were collected at different sizes and microspore development was observed in both light and fluorescence microscopy. A strong positive relationship was detected between flower bud morphology and the specific stages of microspore development. To the author’s knowledge, this is the first report to indicate that the microspore developmental stage can be estimated by flower bud morphology, and applied in the anther/microspore culture of citron watermelon.

Keywords: Drought; Rootstock breeding; Anther culture; Microspore development; Citron watermelon

Introduction

Cucurbita maxima x Cucurbita moschata hybrids are widely used as rootstock for watermelon production because they provide resistance and tolerance to both biotic (especially Fusarium wilt) and abiotic (drought, salinity, and low soil temperature) stress conditions. However, the drought tolerance of these rootstocks is limited and their nematode resistance is not at the desired level [4]. C. lanatus var. citroides (L.H. Bailey Mansf.), which is a wild form in the genus Citrullus, has different names such as “citron”, “citron watermelon”, and “forage watermelon”. It is used in jam, pickles, cooking, water source in arid areas, and pectin for the food industry, in obtaining snacks and oil, and in the medical and cosmetic industry as much as for animalfeed [2,16,19,37]. Due to its high drought tolerance, it is easily grown in arid and semi-arid regions [25]. In our previous studies, two different citron watermelon genotypes were compared to gourd and TZ-148 in terms of yield, phenolic compounds, and antioxidant content, and it has been revealed that they have the potential to be used as a commercial rootstock for watermelon under water stress conditions [31,40].

Citron watermelon is resistant to Meloidogyne incognita [33], and also strains 2 and 3 of Fusarium oxysporum f. sp. niveum have not been reported in watermelon [38]. Citron watermelon is a genetic source against biotic and abiotic stress conditions in watermelon production [5,8,10,15,18,21,26]. They can be crossed with watermelon cultivars (2n=2x=22) and used as candidates in rootstock breeding programs [6]. Its vegetation period is shorter than the winter squash (90-120 days) making it possible to get seeds twice a year [20].

The first step in producing an F1 hybrid variety is the development of pure parental lines and it takes 6-8 generations of inbreeding to reach the desired purity in open-pollinated species such as watermelon. This is a time-consuming, laborious, and high-budget process. Completely 100% homozygous pure lines can be obtained in one generation by anther/microspore culture. Utilization of these techniques allows the production of Doubled Haploid (DH) lines, shortens the process of recombinant pure line development, and allows the creation of high-quality F1 hybrid varieties with DH plants in the early stages of a breeding program. In this way, breeding efficiency is enhanced and the production of new varieties can be accelerated.

As aforementioned, success in anther/microspore culture depends on multiple factors such as microspore development stage, genotype, growing conditions, pretreatments, media composition, incubation conditions, and media refreshment. The microspore development stage is critical for inducing embryogenesis and haploidy frequency. Immature microspores can change direction from gametophytic to sporophytic development (embryogenic re-programming) [32]. It is well known that microspores at the uninucleate stage are generally favorable for the anther culture for many species and genotypes. But it differs among species and genotypes from tetrad, early to late uninucleate to the binucleate stage [3,9,23,27,30,39]. Because of many limitations, the anther culture-based double haploidization process is not generated in citron watermelon as a recalcitrant species.

So, the goals of this study were to compare the shapes of flower buds and the stages of microspore development, as well as to figure out the right size of bud for improving drought-tolerant citron watermelon (Citrullus lanatus var. citroides) rootstocks through androgenesis.

Materials and Methods

Experimental Area, Plant Material, Cultural Practices

The research was carried out in controlled greenhouse conditions. The soil of the research area has a clay-loam structure and contains 2.43% organic substance. It was determined that the EC was 1.05dS/m, the pH was 7.82 and the lime content was 7.3%, and the soil condition was relatively suitable for cultivation. Citron watermelon line SÜ-3 at the S3 level was selected as a donor from the gene pool at the Vegetable Growing and Breeding Department, Faculty of Agriculture, Selcuk University. SÜ-3 has vigorous plant growth habits, oval fruits of medium size, and hard fruit flesh (Figure 1). 2–3 days before planting the seedlings, 175kg/ha of MAP (mono ammonium phosphate) was applied with drip irrigation according to soil analyses. The black plastic mulch was used as a soil cover material to minimize the weed, disease, and pest hazards. The seedlings were planted with 100x60cm row spacing. Conventional cultural practices (disease and pest management, and fertilization) were performed during the entire growing period. Plants were pruned, trained, and developed a single stem morphology.

Figure 1: Plants and fruit of line SÜ-3.

    
    
    Figure 1: Plants and fruit of line SÜ-3.

Collection of Flower Buds

A series of flower buds were collected from healthy and vigorous plants at full flowering time in the morning (7:30–9:00 am). Then, the buds were immediately transported to the laboratory for microscopic observations. The buds were classified based on their sizes ranging from 4.5mm to 18.0mm in width and from 7.50mm to 20.0mm in length and numbered separately (Figure 2 & Table 1).

Figure 2: The classification of bud size in Citron watermelon line SÜ-3.

    
    
    Figure 2: The classification of bud size in Citron watermelon line SÜ-3.

Table 1: Bud classification and size.




  
    No 
    Length (mm) 
    Width (mm) 
  
  
    1 
    7.5 – 9.0 
    4.5 – 5.5 
  
  
    2 
    9.0 – 10.0 
    5.5 – 6.5 
  
  
    3 
    10.0 – 11.0 
    6.5 – 7.5 
  
  
    4 
    11.0 – 12.5 
    7.5 – 9.0 
  
  
    5 
    12.0 – 14.5 
    9.0 – 11.5 
  
  
    6 
    14.5 – 18.0 
    11.5 – 16.0 
  
  
    7 
    18.5 – 20.0 
    16.5 – 18.0



Table 1: Bud classification and size.

Determination of Bud Size, Bud Index Value, Sepal Position, Petal Color, and Microspore Developmental Phase

The length and width of 40 male buds were measured by a binocular microscope, and the average bud size was determined for each bud class. Microspore development was observed in both light and fluorescence-based microscopy (DAPI). For light microscopy analysis, about 50 pollen mother cells from 10 male buds were processed at each specific bud size. Anthers were extracted from buds, stained with 1% acetocarmine solutions for 5 min, and squashed over a microscope slide. Then excessive tissues were discarded, a cover slip was placed over them, and the diameter of the microspores was measured in three different observation fields at 400 (40x10) magnifications. The bud index was calculated separately for each bud examined and expressed as a width/length ratio. The position of the sepal (calyx) relative to the petal (corolla) and petal color were determined by visual observations.

For the fluorescence microscopy, five male buds were processed for each size. Buds were fixed in Carnoy solution (3:1 absolute ethanol and glacial acetic acid) for 24h and then immersed in 1NHCl for 30 min to digest the exine. Anthers were extracted from buds, stained with 1-2 drops of DAPI (4’-6-di amidino-2-phenylindole), and observed under the fluorescent microscope at 400 (40x10) magnifications.

Data Evaluation

The study was carried out in a randomized plot design with 3 replications. The data obtained were subjected to variance analysis in the JMP-13 package program. Significant differences between the applications were determined according to the 5% significance level and interpreted.

Results and Discussion

A limited number of researchers were reported to have produced double haploid lines in watermelon and citron via anther culture. It is well known that immature anthers including the late-uninucleated or early-binucleated phase at the beginning of the first mitotic division are amenable material for the initiation of anther culture for many species. Thus, microspore development (microspore diameter, uninucleated and binucleated phases) and floral morphology (bud length and width, bud index, sepal position) were investigated to identify the appropriate stage for the anther culture in citron watermelon.

We observed 7 distinct bud sizes associated with microspore developmental phases. The intact and autofluorescent-bearing exine was an important factor in preventing the identification of appropriate microspore development phases for anther culture of citron watermelon. Therefore, examinations with the acetocarmine staining technique failed to determine the microspore development stages. A similar bottleneck was reported in cassava [35]. However, the application of HCl, optimized for citron watermelon, provided successful digestion of the exine and facilitated the detection of microspore development stages.

Statistical analyses revealed that there is a strong relationship between microspore diameter, bud size, bud index, sepal position, and petal colour and their corresponding microspore development phase and depicted that bud morphology is an indicator and a convenient parameter to predict the appropriate microspore development stage, and could be used reliably in anther culture of citron watermelon. Similar results were reported for anther/microspore culture in many crops [3,7,17,22,23,34,41].

In our study different bud sizes produced different stages of microspores development. The average microspore diameter increased as the microspore development phase progressed, and reached its maximum size at the mature pollen (late two-celled pollen) phase. Each microspore developmental phase is characterized by a certain bud shape and size (Table 2 & Figure 3).

Figure 3: Flower bud and pollen development in ‘Citron watermelon line SÜ-3’.

    
    
    Figure 3: Flower bud and pollen development in ‘Citron watermelon line SÜ-3’.

Table 2: Bud Class (BC), Microspore Diameter (MD), Bud Size (BS), Bud Index (BI), Sepal Position (SP), Petal Color (PC), and Microspore phase (MS).




  
    BC
    MD (μm)
    BS (mm)
    BI
    SP
    PC
    MS
  
  
    Length
    Width
  
  
    1
    41.24 e
    8.24 e
    5.23 e
    0.633 e
    Above
    Green
    EU
  
  
    2
    46.01 d
    9.47 d
    6.18 e
    0.651 d
    Above
    Green
    LU
  
  
    3
    51.13 c
    10.51 d
    6.94 de
    0.661 d
    Above
    Green
    LU - EB
  
  
    4
    54.96 bc
    11.81 cd
    8.32 d
    0.706 c
    Mostly Equal
    Light Green
    FB
  
  
    5
    57.04 b
    13.56 c
    10.61 cd
    0.782 bc
    Below
    Green-Yellow
    BI
  
  
    6
    60.57 ab
    16.39 ab
    14.09 b
    0.860 a
    Below
    Yellow-Green
    BP
  
  
    7
    63.14 a
    18.81 a
    16.47 a
    0.876 a
    Below
    Mostly Yellow
    BM (OBA)
  
  
     
    LSD: 2.61
    LSD: 2.39
    LSD: 1.97
    LSD: 0.32
     
     
  
  
    EU: Early    Uninucleated; LU: Late    Uninucleated; EB: Early    Binucleated; FB: Binucleated; BI: Binucleated-Immature; BP: Binucleated-Premature; BM: Binucleated-Mature; OBA: One day Before Anthesis



Table 2: Bud Class (BC), Microspore Diameter (MD), Bud Size (BS), Bud Index (BI), Sepal Position (SP), Petal Color (PC), and Microspore phase (MS).

The average diameter of the microspores was measured at 41.24μm, and their green buds were 8.24mm in length and 5.23 mm in width at the Early Uninucleated (EU) phase. The Bud Index (BI) was calculated at 0.633 and the sepal was above the petal. The Late Uninucleated (LU) microspores were 46.01μm in diameter, and their green buds measured 9.47mm in length, 6.18mm in width, and 0.651 BI. The sepal position of buds contained LU microspores above the petal.

The microspores between the LU and Early Binucleate (EB) phases contained both uninucleated and binucleated microspores, mostly uninucleated. It has been determined that the nuclei in the binucleated microspores (with a big vacuole) were quite small and difficult to observe. The microspores between LU-EB were found in buds of 51.13μm in diameter and their sizes were 10.51mm in length and 6.94mm in width. The BI value was determined at 0.661, and the sepal position of green buds was above the petal.

The First Binucleated (FB) microspores were found in relatively light-green and oval-shaped buds with a BI of 0.706. Their length and width were measured at more than 11mm and 8mm, respectively. The diameter of microspores was 54.96μm, and the sepal was mostly equal to the petal. The buds had more than 0.661 BI and contained only two nuclei of pollen.

The diameter of microspores at Binucleated Immature (BI) was 57.04mm. They were clearly and easily observed and had larger (vegetative) and relatively smaller (generative) nuclei. The buds were green-yellow and their petal was prominently longer than their sepal. They were 13.56μm in length and 10.61μm in width. The microspores and the buds continued to grow and develop. The Binucleated-Premature (BP) pollens were detected in yellow-green and relatively globe-shaped buds. They had 16.39mm length and 14.09mm width, with a shorter sepal (nearly 50% of the petal) and 0.860 BI. The average diameter of the microspores was measured at 60.57μm.

Binucleated-Mature (BM) pollen grains had 63.14μm diameter and visible quite large two nuclei with a big vacuole. Its bud shapes were nearly globe-shaped with a BI of 0.876, and buds were 18.81mm and 16.47mm in length and width, respectively. The color of the petals was predominantly yellow, with an easily visible green vessel. The sepals were quite small, hardly visible, and sometimes deformed. The average size of citron microspores and pollen grains is considerably narrower than squash, pumpkin, and winter squash [13] but similar to melon [23].

A positive correlation was found between flower-bud size (length and diameter) and microspore development stages in the carob tree [7], cassava [35], alfalfa [41], and melon [24]. On the other hand, the correlation (R²=0.59) between bud size, anther length, and microspore development stages showed that anther length was a reliable indicator in the tomato [3]. Contrary to the findings of Adhikari and Kang (2017), a considerable amount of publications suggested that anther sizes should not be used as a parameter in determining the appropriate microspore development stage in tomato [30], eggplant [28] and melon [24].

Our findings indicated that the buds were 5.0-6.0mm in width and 8.0-9.5mm in length and contained only uninucleated microspores. The microspores between uninucleated and binucleated were obtained from buds that measured 7.0mm in width, and 10.5mm in length. These stages have been identified as a characteristic and critical point for success in the anther-based double haploidization process of citron watermelon. Hence, the anther that contained pollen at the uninucleated stage shows a better response and uninucleated pollens were observed in flower buds that had 4.5-5.0mm length in melon [11]. The middle to late uninucleated microspore stages was the best in anther culture of winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) [13]. In Lagenaria siceraria (Molina) Standl, pollen at the uninucleated stage collected from 5-7mm buds, is the most appropriate for androgenesis [12]. Male flower buds (10-12mm in length) containing microspores at the late-uninucleated stage are the best material for haploidization in watermelon [1,29].

The sepal (calyx) position of green buds contained microspores from uninucleated to uni-binucleated was longer than petal (corolla). The buds with mostly equal petals and sepals contained binucleated microspores in citron watermelon. Besides, Wang et al. (2015) argued that the microspore development stage was closely related to bud size and anther color, the petals were calyx slightly and the anthers were yellow and contained productive microspores at late-uninucleated in melon. On the other hand, the visual parts of the sepal and petal were a less reliable morphological feature in tomatoes [23].

Conclusion

Breeding of new cultivars and rootstocks tolerant to abiotic (drought and salinity) stress factors could be more profitable than exogenous applications in sustainable agriculture. Double Haploidization (DH) is a unique process to obtain pure lines that form the basis of F1 hybrid cultivar breeding programs. Anther culture is a way for the induction and development of microspore-derived plants in many species. It’s well known that, this technique is complex and its efficiency is affected by many factors. Success in anther culture-based DH is considerably associated with the culture of microspores at an appropriate developmental stage. A close relationship was observed between bud morphology and the specific stages of microspore development. It could be safely used for the selection of an appropriate bud size containing the best development stage of microspore and applied in the anther/microspore culture of citron watermelon as a recalcitrant species. However, our findings established a citron watermelon line, to generalize these findings; it would be beneficial to study with a large number of genotypes.

Acknowledgments

This study was funded by the Scientific and Technological Research Council of Turkey (TÜBITAK-1001) (Project No: 221O338).

References

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Citation:Kurtar ES, Seymen M, Alan AR, Toprak FC, Atakul Z, et al. Determination of the Microspore Development Concerning Floral Morphology for Improving Drought Tolerant Citron Watermelon (Citrullus lanatus var. citroides) Rootstocks Via Androgenesis. Ann Agric Crop Sci. 2023; 8(2): 1130.

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