Induction of Microspore Embryogenesis of Eggplant (Solanum melongena L.) ‘Gelatik’

ABSTRACT The haploid or double haploid plant of eggplants could be produced from microspore culture (embryogenesis of microspores). In the breeding programs, microspore can be developed into an embryo directly after exposure to stress treatment during cultured. Stress (temperature and starvation medium) is an important factor in the induction of embryogenesis microspore. This study aims to induced embryogenic microspores from eggplant CV. Gelatik. The stage late-uninucleate microspore (Vacuolate Microspore/VM) and early binucleate (Young Bicellular Pollen/YBP) are the suitable stages to induce multinucleate structure. There are 3 methods used in this research; 1) Determination of the stage development of microspore based on flower buds length and anther length. 2) Induction of embryogenic microspore on the pre-treatment and starvation medium. 3) After giving pre-treatment for 4 days, micropores were transferred to culture medium A2 at 28oC in dark conditions to induce the multicellular structures. This study reported that 50-68.51% of the VM+YBP stage obtained in the range of flower bud lengths of 10-17 mm, and 5.06.9 mm, the range of anther length containing VM+YBP of 50-77.48%. The pre-treatment heat shock at 33oC in the medium B for 2 days, produced embryogenic microspores with a high percentage, that is about 50.19%, while microspores at 25oC and 4oC respectively 46.17% and 49.28%. Pre-treatment for 4 days at 4 oC, 25 oC, and 33oC with the percentage of embryogenic microspores apiece 32.87%, 27.45%, and 37.34%. The multicellular (starlike) structure begins forming on the fifth day of incubation in culture medium (A2) after pre-treatment in B medium at 33oC.


INTRODUCTION
Eggplant (Solanum melongena L.) is one of the essential vegetables in tropical and subtropical regions around the world. It is the fifth most economically crucial solanaceous plant after potatoes, tomatoes, pepper, and tobacco (FAO, 2014;Taher et al., 2017). Improvement on eggplant production carried out through biotechnology and hybridization approaches (Kalloo, 1993;Kashyap et al., 2003;Magioli and Mansur, 2005;Bal et al., 2009) such as regeneration of haploid or double haploid plants, through anther or microspore culture (Microspore Embryogenesis). Microspore embryogenesis represents a unique system which can produce homozygous lines in a relatively short period and does not require much effort and cost compared to conventional crossing methods which require a lot of crossing over time (Snape, 1989;Maluszynski et al., 2003;Adhikari and Kang, 2017). Usually, microspores will develop into mature pollen (pollen grain) and ready to fertilize an egg. Through in vitro culture, the normal development of microspores can be transformed into the development of the sporophytic phase that will form embryos directly. The ability of male gametophytes to change their developmental fate from pollen to embryonic development can occur when exposure to stress treatments during culture. This process referred to as microspore embryogenesis (Soriano et al., 2013). Stress treatment has an essential role in this process, encouraging microspore differentiation and conditioning the androgenic responses (Munoz-Amatriain et al.,

2009).
In microspore embryogenesis, free microspores isolated from anther are cultured in vitro and after pre-treatment stress, microspores will develop sporophytically (Shariatpanahi et al., 2006;Bal et al., 2009). Some stress treatments have been known to induce embryogenic microspores, including cold shock, heat shock, carbon starvation, and nitrogen in growing medium and colchicine treatment (Moraes et al., 2004). During the embryogenesis induction, identification of microspores that have embryogenic abilities and the mechanism of transformation of microspores into embryogenic cells are carried out. Although the initial stage of development of microspores for the induction of embryogenesis differs between species, usually microspores are chosen in the vacuolated stage. Vacuolated microspore stage is the development stage that allows reprogramming of microspores in most species (Olmedilla, 2010). Salas et al. (2012) revealed microspores at the stage of young microspore (YM) and mid-microspore (MM) in eggplant did not show a growth response after being transferred to embryogenesis medium. The optimal stage for embryogenesis induction is not the transition stage from vacuolate microspore (VM) to the young bicellular pollen (YBP) stage. When VM and YBP were cultured in a liquid medium, both showed a growth response towards embryogenic microspores. In this study, we determine the stage of microspore development through the selection of flower buds as the first step in microspore culture. To observe the anatomy of the anther, we analyze the correlation between the stages of microspore development and the stage of anther development. Furthermore, induction of embryogenic microspores by pre-treatment of temperature stress (4 o C, 25 o C, and 33 o C) with a combination of medium starvation (B) for 4 days will follow, the protocol of the microspore culture of eggplant cv. Bambino (Bal et al., 2009).

Plant Material
The seeds of eggplant cv. Gelatik were sown in a plug-tray and transferred to a pot in a greenhouse of Laboratory of Biotechnology, Faculty of Biology, Universitas Gadjah Mada. The seedling, one and a half months of age or has 4 leaves transferred to a pot measuring 30x27x17 cm 3 and filled with planting media consisting of soil, manure, and compost. The plants watered daily, and the soil was supplemented with Growmore fertilizer NPK (15:15:15) and for leaves.. The plants were maintained and pruned by bud shoot (dominancy apical) to produce many branches. Flower buds then picked as the primary materials of this study.

Determination of Microspore Developmental Stage
The flower buds of various sizes were collected and grouped according to the size of the flower bud length and anther length (shown in Table 1). The buds were measured using Nankai digital calipers 0-150 mm. The length measured from the base of the calyx to the tips. After the measurement, the anther removed from the flower bud. Anther from each flower bud was crushed in medium B to observe the development of microspores by Microscope (Nikon Diaphot 300 Fluorescence; Japan) and images were taken by OptiLab face 2.2 Miconos. Determination of the percentage of each microspore stage in the same anther was counted from 10 randomly selected images from five display fields. The number of each microspore is calculated by dividing the number of microspores at a certain stage by the total number of microspores based on the calculation of 10 images.

Induction of Microspore Embryogenic with Temperature Stress and Starvation Medium
Anther from flower buds contained high-percentage VM + YBP microspores were used as microspores donors for the induction of embryogenic microspores by stress treatment. Microspores were cultured in B Medium (Indrianto et al., 2014) containing macronutrients: KCl 1.490 mg/L, MgSO 4 .7H 2 O 250 mg / L, CaCl 2 .2H 2 O 110 mg / L, KH 2 PO 4 136 mg / L, Mannitol 54.630 mg / L with pH 7. The cultures incubated at 33 o C, 25 o C, and 4 o C for 4 days. The observations were presented as a percentage of embryogenic microspores (VM, YBP, and Multinucleates) and non-embryogenic (YM, MM, and lysis microspores).

Statistical Analysis
The quantitative data consists of the number of embryogenic microspores and the increased diameter of microspores at different temperature treatments were analyzed using Microsoft Excel 2019. The data were obtained from three replications and presented as mean ± standard deviation in the tables.

Developmental Stage of Microspore of Eggplant cv. Gelatik
The suitable stage of the flower bud ( Figure 1) is a critical determinant of the success of microspores culture. The morphological size of flower buds, the length of flower buds, and the length of anther correlate with the stage of microspore development and the stage of anther development. In some studies, bud length and anther length are also used as benchmarks for the stages of microspore development such as in anther culture of tomato (Segui-Simarro and Nuez, 2005), eggplant (Salas et al., 2012), and correlation with the development of pollen in anther culture of apple (Zhang et al., 2013).
In this study, the microspore development stages were identified based on the length of flower buds and the length of anther of eggplant cv. Gelatik. Table 1 shows that the VM + YBP stage of 50-68.51% is in the range of flower bud length of 10 -17 mm, and 5.0-6.9 mm range of anther length containing VM + YBP of 50-77.48%. The highest percentage of YM + MM was observed in the range of flower bud length and anther length of 8.0-9.0 mm with a percentage of about 66.54% and 4.0-4.9 mm at 66.02%. The length of anthers was suitable for cultured in the starvation medium at 5.0 -5.9 mm, not for all measures. In this study, the same flower bud length has a different anther length. Therefore the percentage of mature pollen in the range of flower bud length 14-15 mm is lower than 16-17 mm or 12-13 mm. The YM and MM after pretreatment at 33 o C, 25 o C, and 4 o C, then subcultured into A2 medium did not show a growth response, even some microspores experienced shrinkage. Salas et al. (2012) also stated that in the microspore culture of eggplant cv. Bandera. In the study of morphological markers for the development of microspores in maize, Moraes et al. (2008) stated that anther length was the most reflected cytological stage of microsporogenesis. In wheat, it is also found that the length of the anther is considered an appropriate morphological marker for the assessment of the specific development of microspores (Immonean and Antila, 1998). However, it is essential to recognize that the measurements obtained for these parameters in each microspore developmental stage varied according to the genotype and the cultivation place.

Developmental Stage of Eggplant Anther
Stamens consist of two different structures, namely anther and filament. The length of the anther indicates the stage of anther development and the stage of microspores in the anther locus. Microspore development stages were identified based on cellular characteristics such as cell shape, cell number, nucleus type, and position in the cell, and state of the chromosomes (Adhikari and Kang, 2017). According to Browne et al. (2018) the length of the wheat anther is an accurate parameter measure to determine the stage of anther development and can be applied among wheat cultivars. Each stamen contains filaments and anthers with four lobes connected to the filament by connective tissues (Zhang and Wilson, 2009). In this study, the stage of anther development of wheat (Browne et al., 2018), used as a reference for determining the developmental stage of the anther in eggplant cv. Gelatik. In this research, the observation of anther development is starting from stage 8 ( Figure 1A), namely the Programmed Cell Death (PCD) tapetum stage and the last tetrad stage (before the microspore tetrad is released as young microspore). Stage 9 anther ( Figure 1B), which is the stage in which tetrad microspores are released as young microspores. Stage 10 ( Figures 1C, 1D, 1E) is the vacuolated microspore stage. Stage 11 ( Figure 1F), the anther contains the early binucleate microspores. Stage 12 ( Figure 1G), the tapetum portion is completely degraded. Stage 13 ( Figure 1H), the septum between the upper and lower locus begins to degrade. Stage 14 ( Figure 1H), initial dehiscence, and stomium are degraded. The last stage of anther development is stage 15 (Figure 1I), where pollen released from the anther locus.

Induction of Embryogenic Microspore
The range of flower bud lengths 10-11, 12-13, 14-15 and 16-17 mm each contains VM + YBP 59.07%, 68.51%, 54%, and 56.03. The size of the flower bud used 12-13 mm for embryogenic microspores. Another main factor besides the phase of flower buds is the stress treatment because this is the main factor to determine the success of microspore embryogenesis induction. Microspores were cultured in 2 ml of liquid B medium for 4 days and incubated at three different temperatures (4 o C, 25 o C, and 33 o C). The parameters observed were the percentage of embryogenic microspores (late uninucleate, binucleate and multinucleate microspores), and nonembryogenic microspores including young microspores (YM), mid-uninucleate (MM) and lysis or shrinking microspores. Microspores at this stage, if exposed to stress treatments (such as heat shock, cold shock, and starvation medium) will develop into an embryo. A high percentage of embryogenic microspores is also a determinant success of the stress treatment used. The results showed ( Table 2) that, embryogenic microspores were observed during pre-treatment at 33 o C (heat shock), 25 o C and 4 o C (cold shock) for 2 days of culture and liquid B medium, respectively 50.19%, 46.17%, and 49.28%, after 4 days of incubation obtained embryogenic microspores of 32.87%, 27.45% and 37.34% at each incubation temperature. After 4 days of incubation, several microspores were lysis and shrinking. Incubation microspore of eggplant cv. Gelatik at 33 o C for 2 days, enough to induce embryogenic microspore. This is different from broccoli when the embryo production is significantly increased in almost all genotypes of broccoli, which is incubated in a medium of ½ NLN-13 at 32 o C for 1 day, compare to if it is incubated at a standard temperature of 30 o C for 2 days. Bal et al. (2009)  and starvation were the same as Miyoshi's (1996) study, which cultured eggplant microspores in mannitol starvation medium and incubated at 35 o C for 3 days. The incubation for 48h at 32 o C obtained unoptimized results for embryo production. That research reported that broccoli is sensitive to high temperatures (Carlos and Dias, 2001). In eggplant cv. Bambino, microspores treated with cold shock rapidly will lose viability after being subcultured into an AT3 medium (Bal et al., 2009). The same problem was also found in this study, the pre-treatment incubation of microspores at 4 o C for 4 days after subculture to A2 medium were found many microspores underwent lysis. Microspores that underwent nucleus symmetrical division and form multinucleate structures were obtained at low frequency after the microspores were transferred from heat shock and starvation to the AT3 medium containing 0.25 M maltose. The frequency of multinucleate structures were 17.3% and 19.4% after one week of incubated in an AT3 medium (Bal et al., 2009). Table 3. showed, the late uninucleate and early binucleate microspores were cultured in liquid B medium and incubated at 33 o C for 4 days. There is an increase in the diameter size of microspores, respectively about 2.759 μm and 3.745 μm. Late uninucleate and binucleate culture of the microspores at 25 o C for 4 days was 1.6 μm and 2.285 μm respectively, whereas microspores were at 4 o C for 4 days each at 0.421 μm and 1.125 μm. Microspores incubated at 4 o C developed very slowly. The increase in the diameter size of the microspore of both the late uninucleate and binucleate is unlike if the microspores incubated at temperatures 25 o C and 33 o C. According to Bal et al. (2009), heat shock was successfully used as a stress factor for microspore embryogenesis in eggplants. However, starvation pre-treatment, combined with heat shock, namely the microspore embryogenesis protocol in tobacco, is more effective. Stress treatments such as heat shock also provide optimal results for the induction of embryogenic microspores in eggplant cv. Gelatik. In addition to temperature stress, incubation time is also important in the induction of embryogenic microspores in eggplant.

Development of Microspore Embryogenic in Culture Medium
Embryogenic microspores have been treated with heat and cold shock temperature stress for 4 days, then subcultured into embryogenesis medium A2 to release microspores from stress to develop microspores towards embryo formation. Embryogenic microspores from B medium at 33 o C ( Figure 2) after one week of incubation in a new medium (medium A2), swoll very quickly, while in embryogenic microspores from 25 o C to 25 o C, many microspores were lysed. Thus microspores from 4 o C temperature were more lysed with very slow development and the number of embryogenic and starlike microspores was less than the others.
Embryogenic microspores formed after stress treatment were characterized by several physiological and morphological changes (Figure 2). In tobacco and wheat, embryogenic microspores form a nucleus in the middle surrounded by structures like vacuoles. Microspores with cytoplasm form starlike structures, commonly found in microspores of wheat, barley, and tobacco. Swollen in Brassica is marked as embryogenic microspores (Bal et al., 2012). In this study, several embryogenic microspores were observed, swollen, increased diameter of the microspores, the cell nucleus continuously divided to form multinucleate structures. The induction of microspores embryogenesis in eggplants in A2 medium (Figure 2), after 3 weeks of incubation produced no globular embryo, only microspore enlargement or bulging into swollen microspore, and  only formed a star-like structure. This shows that the A2 medium is less suitable for eggplant embryogenesis. According to Sumarmi et al. (2014) A2 medium is more suitable for the microspore culture of monocot plants. Medium A2 succeeded in spurring the growth of embryogenic microspores, resulting in symmetrical division and multinucleate structures formed in the culture of palm microspores (Indrianto et al., 2014). Besides the A2 medium, some embryogenesis medium often used for induction of embryogenesis is AT3 medium in the culture of eggplant cv. Bambino (Bal et al., 2009), and B5 medium with 9% maltose in wheat microspore culture (Zheng, 2003).

CONCLUSION
The incubation of microspores at 33 o C in the medium for 4 days is effective to induce embryogenic microspores in eggplants. In addition, the early binucleate stage is preferably chosen for the induction of embryogenic microspores.