Effect of pollination method on true shallot seed formation improvement

Producing seeds from shallot flower requires proper pollination techniques to form pithy seeds. This study aims to determine the effectiveness of several pollination methods in producing true shallot seeds (TSS). The research was conducted in Cilame Village, Ngamprah District, West Bandung Regency, West Java at an altitude of 900 m asl. The study used a completely randomized design (CRD) which consisted of four treatments and six replications. The treatments are pollination methods: control (natural pollination), using a brush, using stroking/bare hand method, and a combination of using a brush and stroking. Pollination treatment was carried out when the flowers have bloomed for three consecutive days. The variables observed were plant height, number of tillers, number of flowers per clump, umbel diameter, number of capsules per umbel, and seed production per umbel. Data were analyzed using ANOVA, and if the results of the F test were significantly different, it was continued with DMRT (Duncan’s Multiple Range Test) and correlation. The result showed that pollination by stroking is the most effective to increasing TSS production per umbel. Seed production per umbel by stroking was 0.89g and control (natural pollination) was 0.23g. Stroking method increased TSS production per umbel by 74.16 % compared to control. Therefore, the method could become an alternative to increase the formation of TSS in overcoming the limitations of natural pollinator.


Introduction
Shallots can be propagated by two types of seeds, namely True Seed of Shallot (TSS) and seed bulbs.As a planting material, TSS has more advantages than seed bulbs because TSS is free from seed-borne diseases that have the potential to reduce pesticide applications, has higher productivity, smaller quantity making it easier to transport, and has a longer shelf life [1][2][3] The production of TSS is determined by various yield components [4], such as the number and weight of umbels, number of capsules, bulb weight, and total plant weight.Translocation of assimilating materials to sinks is important for seed development.The sink and source ratio describes the distribution of assimilated materials and nutrients during plant growth [5].The formation and filling of seeds can be limited by the availability or use of assimilation which is called the source or sink limit [6].But before that, pollination holds the gate to seed succession.Pollination can be done naturally such as by wind and water (abiotic) or with the help of pollinators such as insects (biotic) [7].The function and number of pollinators in an area is affected by environmental changes and intensive land use.In a rotational cropping system, pollinating insect species become numerous due to the availability of various food sources (plants) [8].Over large areas, pollinator species richness is positively related to the availability of sugar nectar in flowering plants [9].In addition, pollination is influenced by plant physiology and ecology and the characteristics of pollinating insects [10].For insect pollination to occur, the flower must be in the right place to reflect sunlight onto the pollinator, so that the pollinator can find the flower.
The success of pollination can be seen from the formation of capsules (fruit sets) and seeds.The percentage of capsule formation is greater with the help of pollinating insects (cross-pollination) compared to other methods because shallots are dichogamous and partly self-incompatible [11].Among the pollinating insects that are most effective in shallots are Apis cerana and Apis mellifera [12].However, their unexpected results couldn't be the same as what humans could do on purpose.By using bare hands or other tools such as brushes, humans can carry out artificial pollination.
Pollination by hand can maintain the quality of crop production better than natural pollinators.Unfortunately, it also carries risks such as endangering pollinator conservation, resulting in high food prices because it requires a lot of labor, and can lead to over-pollination (fruit malformation).So the benefits of hand pollination must be optimized to offset these risks [13].
Hand pollination can be done on both large and small-scale farms, home gardens and greenhouses.The result depends on the origin of the pollen and its quantity, the timing and frequency of pollination, and environmental fluctuations.Hand pollination has been carried out on oil palm, tomato and apple plants with different considerations.In oil palm due to lack of natural pollinators, in tomatoes due to low efficiency of honeybees and in apples during unfavorable weather conditions.Thus, hand pollination is necessary due to lack of natural insect pollinators, adverse weather conditions and pest infestation [13].
The pollination method with brush can also be applied to shallots as has been applied to strawberries [14] and kiwifruit [15].The disadvantage of using a brush for pollination is that the flower receptacle can be damaged which can lead to lower pollination success rates and lower seed quality [14].Regarding the advantages and disadvantages of the three possible pollination methods applied to shallots, it is interesting to know the results of these three methods and their combination in an experiment.Therefore, the purpose of this study was to determine the effectiveness of several pollination methods in producing TSS.

Materials and Methods
The research was conducted in Cilame Village, Ngamprah District, West Bandung Regency, West Java, at an altitude of 900 m above sea level.At this altitude the percentage of shallot umbels can be optimized because the temperature (<18 o C) supports the generative phase [16].The study used a Randomized Complete Block Design (RCBD) which consisted of four pollination methods and four replications.The number of samples per unit was three plants.The four treatments are: control (natural pollination), using a brush, by stroking, and a combination of using brush and stroking.Treatment is carried out when the flowers bloom.
Seeds used in this research were Bima Brebes.The preparation consists of vernalization, growth regulator treatment, and fungicide.Vernalization was carried out in cold storage at 10 o C for four weeks.This temperature and storage time are known as shallots required for flower initiation [17].The growth regulator used in this study was benzyl amino purines (BAP), which has been shown to be effective in increasing flowering and the amount of capsule formation [18].Fungicides with the active ingredient mancozeb are also applied as needed.
Simultaneously with the preparation of the seeds, land preparation was carried out by applying manure and lime.The width of the beds is 1.2 m and the length depends on the farmer's plot.After the application of 20 tons of manure per hectare, the beds are covered with silver black plastic mulch.The spacing used is 20 x 20 cm, so there are 5 rows of plants.The fertilizer used was NPK 16:16:16, added 10 times at a dose of 60 kg ha -1 , white KCl 10 times at a dose of 2 gr L -1 , and boron fertilizer 3 kg ha -1 at 3, 5, and 7 Weeks After Planting (WAP).The variables observed were plant height (cm), number of tillers (stems), umbel diameter (cm), number of flowers per clump (flowers), number of capsules per umbel (capsule), and seed production per umbel (g).
Observational data were analyzed statistically with ANOVA, using the F test at the 5% level to determine the effect between treatments.If there was a significant effect, then a follow-up test was carried out with Duncan's Multiple Range Test (DMRT) and a correlation test at the 5% level.Data processing used SAS version 9.4.

Plant growth
Factors Plant height and number of shallot tillers at the time of pollination treatment did not show a significant difference (Table 1).This uniformity is an ideal condition for plant samples before treatment.This will produce accurate and optimal comparison data because it can reduce interfering factors that can affect research results [19].6,04 18.49 Note: Numbers followed by the same letter in the same column show no significant difference in the 5% DMRT test.CV of number of tillers was transform with root transformation (x+0.5) Table 2 shows the number of flowers per clump, umbel diameter, number of capsules per umbel and seed production per umbel from all treatments.There were no significant differences in the number of flowers and seed production.However, the diameter of the umbel and the number of capsules suggests otherwise.The combination of stroking and brush pollination methods produced the widest umbel diameter and the highest number of capsules, although it was not significant with the stroking and control methods, respectively.

CV of number of flowers per clump was transform with root transformation (x+0.5), CV of number of capsules was transform with log transformation (log+1) and CV of seed production per umbel was transform with root transformation (x+1)
The formation of the umbel is one of the important stages in the production of shallot seeds.It is the beginning of the generative phase [20] where the process of forming generative organs, flower differentiation, maturation of floral parts, anthesis, and initiation of flowering will occur.It is a morphological transition from vegetative shoots to generative shoots which can be detected from changes in the shape and size of the shoots [21].Umbels with wider diameters tend to have more capsules per umbel.A large number of capsules per umbel has the potential to produce more seeds per umbel if the seed formation process goes well and perfectly.The results of research [22] on the Trisula variety showed that the greater the number of flowers and capsules produced, the more seeds were produced.However, this scenario does not apply to the results of this study.
The Stroking and brush pollination method had the largest umbel diameter and the highest number of capsules per umbel but did not have the highest seed production per umbel.This means that the percentage of seeds formed in the capsule is small, presumably due to the lack of contact with human skin.As can be seen in table 2, the brush method and natural pollination also have a low percentage of seed production.Seed production per umbel by stroking was 0.89g and control (natural pollination) was 0.23 g.Pollination by stroking had the highest seed production per umbel although the number of capsules per umbel was less than the combined method.Stroking pollination increased TSS production per umbel by 74.16% compared to control (natural pollination).It was optimizing the formation of pithy seeds in capsules/umbels.Pollination assisted by Stroking and or brush has a great opportunity to speed up the fertilization process.However, using a brush can cause damage to the flowers as there is a greater chance of the process being more frequent and rougher than it should be.
Factors that affect the increase in the number and quality of seeds through stroking method is due to direct pollen crossing, whereas natural pollination usually brings a mixture of pollen and crosses itself on flowers so that the success rate of seed formation is low.Although Insect-assisted pollination could increase seed yield by an average of between 18 -71%, the success of insect on a plant pollination so determined by the uncertain factors such as the level of visits and the number of pollinating insects around the plant [23], the characteristics of flowers as a major attraction for pollinators [24], and environmental changes [8].
An increase in the number of flowers is not always followed by high seed production [25] because a flower consists of many capsules which do not always contain seeds.Some capsules may be empty.Empty capsules are caused by the limited pollen attached to the surface of the stigma, so the number of seeds that develop in one capsule is also very limited [12].The number of capsules and seeds per umbel tends to depend on the activity of pollinating insects [26].In areas that are not a common habitat for pollinating insects, there is a greater chance of having these empty capsules.Under these circumstances, successful fertilization can be replaced by hand pollination [27].
Hand pollination by stroking results in the highest chance of successful pollination.It resulted in the highest value of shallot seed quality compared to other pollination methods [28].It influences other plant species, such as Primula secundiflora, to increase flower number, crown tube length, crown tube entrance diameter, number of fruits, viable seeds per fruit, and female fitness (total viable seeds per individual) [29].It also resulted in better quality strawberry seeds [14] and increased the number of flowers and cocoa yields by 161.5% and 51.3%, respectively [30].

Correlation growth, yield components, and true seed of shallot production
There was a significant positive relationship between plant height and the number of capsules per umbel, and between umbel diameter and the number of capsules per umbel.This means that the taller the plant grows or the wider the diameter of the umbel, the more the number of capsules per umbel (table 3).Although in this study the number of capsules also correlated positively with the number of seeds formed or seed set, this relationship was not significant.This means that the more capsules are formed, the more seeds are formed [31] but some capsules cannot produce pithy seeds or some did not have seeds.
Low capsule and seed formation can be caused by improper pollination time between mature pollen and receptive stigma, which will affect the TSS quality [12].Pollination success is also determined by the anthesis time.Pollination will be accurate and increase the success ratio of fertilization when pollen and pistils are at their optimum performance point [32].
The success of pollination depends on the reception of the stigma, and the effectiveness of the pollinator's visit to the receptive stigma.Receptive stigmas allow adhesion of pollen grains which can be followed by hydration of the pollen grains.The reproductive process that occurs is determined by the amount of pollen carried by the pollinator and the interaction of the pistil with the pollen [23].So, a pollination method has a major factor on the production of TSS.

Conclusion
Hand pollination by stroking is the most effective for increasing TSS production per umbel.Seed production per umbel by stroking was 0.89g and control (natural pollination) was 0.23 g.Stroking method increased TSS production per umbel by 74.16% compared to control.This pollination method could become an alternative to increase the formation of TSS in overcoming the limitations of natural pollinators.

Table 1 .
The growth of shallots by various methods of pollination on the production of the true seed of shallot in Ngamprah District, West Bandung Regency, West Java.

Table 2 .
Yield components with several methods of pollination on the production of the true seed of

Table 3 .
Correlation between growth, yield components, and true seed of shallot production in Ngamprah District, West Bandung Regency, West Java