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

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: ertansaitkurtar@selcuk.edu.tr

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.