Observation on local high-yielding stevia (Stevia rebaudiana Bertoni L.) clone specific to Bandung District, West Java

The use of stevia (Stevia rebaudiana Bertoni L.) as a natural sweetener shows great potential. However, in Indonesia, the lack of superior and highly productive varieties has been a major challenge in its development. Nukita Co-op has developed a promising stevia clone which we evaluated in this study for its readiness to be proposed as a new and improved variety. Our research was conducted in August 2023 through primary and secondary data collection. Primary data comprised the clone development process and morphological variations observed in farmers’ fields, while secondary data included crop productivity and types of stevia-based products made. Our findings revealed that the CM3 clone, derived from a Vietnamese variety, has been cultivated by farmers in Bandung district since 2010. It has high yields (up to 7-ton dry leaves/ha/year) and is marketed as simplisia (unprocessed ingredients) and various tea variants. However, we noted high morphological variations in farmers’ fields. Therefore, stevia clone CM3 is not ready for direct release. With breeding efforts, this clone could be improved through the selection process and released as a high-yielding variety to support stevia’s development as an alternative sweetener.


Introduction
Sugar is considered one of the twelve essential food commodities that the government must protect and guarantee [1].Generally, people consume sugar for its natural sweetness in foods and beverages, as well as for its preservative properties [2][3][4].The Sugarcane plant is a popular source of natural sweeteners, with Indonesia alone consuming an average of 5.7 million tons of sugar per year.However, the national sugar production only reaches 2.5 million tons, resulting in a deficit of 3.2 million tons that must be imported [4].Over the past five years, the growth rate of sugar imports has been higher than that of consumption, productivity, and production.Despite this, the natural sources of sugar have not been able to meet the ever-increasing demand.
Many manufacturers in the food and beverage industry opt for synthetic sweeteners to cut down on production expenses.However, it's important to note that the two most used synthetic ingredientscyclamate and saccharine -are carcinogenic [5].Furthermore, excessive sugar consumption has been linked to health issues such as obesity, diabetes, and other complications associated with these two diseases, which tend to affect the middle and upper classes more.As a result, there are ongoing efforts to find natural sweeteners that are low in calories and safe for consumption [6].
Numerous studies, both domestic and foreign, have demonstrated the potential of stevia as an alternative source of sweeteners [6][7][8][9].Stevia leaf extract contains steviol glycoside as its primary active ingredient, with stevioside being the most abundant component.Unlike saccharine or aspartame, stevioside is resistant to damage at high temperatures (200°C) [10].Additionally, stevioside is 300 times sweeter than sucrose [11], non-caloric, and does not have any addictive or obesity-causing effects.Therefore, it is safe and suitable for consumption by individuals with diabetes, obesity, or sensitivity to synthetic sweeteners, as well as for general health.
Stevia leaves contain non-toxic and anti-mutagenic compounds that have numerous benefits, including antihyperglycemic, antihypertensive, antioxidant, antibacterial, and antimicrobial properties [12].These antibacterial properties can inhibit the growth of bacteria that cause tooth decay.Additionally, compounds such as alkaloids, tannins, and flavonoids found in stevia leaves are antiplaque and can be used as a remedy [13][14].With these advantages, stevia sweeteners have great potential as an alternative to imported sugar and even as an export market with endless possibilities.
Developing sweetener plants, such as stevia, in Indonesia faces a major obstacle due to the absence or scarcity of superior varieties that can produce high-quality and high-yielding crops [15][16][17][18][19].The Agricultural Biotechnology Research Institute of Indonesia (formerly known as the Agriculture Research Institute or BPP) has been working to create superior stevia varieties since 1984.They have introduced the BPP series of stevia varieties that can produce approximately 2.5-3.5 tons of dry leaves per hectare per year, with stevioside content ranging from 6.2-15.1% and A reb A 0.4-3.7%[9].However, this productivity is relatively low compared to foreign stevia varieties.For instance, in the US, superior varieties can generate more than 5 tons of dry leaves per hectare per year, with a total steviol glycoside content (TSG) of 15% and reb A of 9% [9].Moreover, there are claims of new varieties that can produce a TSG of 21% with reb A 16% [9; 20-21].The Agricultural Biotechnology Research Chamber was unable to locate the superior clone among the BPP series stevia varieties, including BPP 72.Currently, local farmers' stevia crops are of uncertain legality and are often impure due to mixed cultivation methods.As a result, these plants have low productivity and quality, which does not meet the desired demand or quality standards.Additionally, it is believed that these stevia clones leave a bitter aftertaste due to high stevioside content and low reb-A content [22].
To tackle the obstacles mentioned above, an attempt has been made to cultivate stevia strains that are both high-yielding and attractive to consumers.These endeavours encompass a range of techniques, such as exploring and integrating genetic variation, as well as utilizing conventional hybridization, invitro breeding, and ploidization [23][24][25].The outcome of these endeavors has yielded stevia clones that boast high productivity, including a local creation from Nukita Co-op, named CM3.This study seeks to assess the viability of this CM3 clone as a potential stevia variety.

Methods
The research was conducted in August 2023.The location study was selected purposively, that is, the CM3 development areas in Bandung District, West Java.To evaluate whether the CM3 clone is ready to be released as a superior stevia variety, we measured two types of data components: primary and secondary.These include the clone's origin and development process, data on its superiority in terms of yield, quality, or other important traits, and most importantly, the clone's uniformity and stability performance.
The National Research and Innovation Agency (BRIN) sent a team of stevia breeders to visit the Nukita Co-ops office in Bandung.The team interviewed the Co-ops manager and her team to gather information about the origin and development process of CM3.We discussed yield data, the economy, and the stevia products that have been developed so far.The team also collected secondary data on CM3 from journals, news sources, and personal communications.
After the interview, the team, accompanied by a local Estate officer, visited the stevia development area to assess morphological variation (primary data).They selected stevia grown by the Farmer group Tani Jayagiri, Lembang, West Bandung district for the study.The team noted general performance and morphological variation based on stevia guidelines produced by the Ministry of Agriculture.
The collected primary data obtained were then analyzed using the descriptive method, whereas the secondary data were used as such.

Results and Discussion
To determine if the CM3 clone is ready to be released, as a superior stevia variety, several components must be assessed.These include the clone's origin and development process, data on its superiority in terms of yield, quality, or other important traits, and most importantly, the clone's uniformity and stability performance.Once all these factors have been evaluated, a decision can be made whether we will use it directly for variety or selection efforts are still required to improve its performance to fulfil the requirement for release variety.

Origin and development process of stevia CM3 clones
The development of stevia CM3 was meticulously researched through conducting direct interviews with breeders from the National Research and Innovation Agency (NRI).The NRI team paid a visit to the Nukita Co-op office in Bandung where they engaged in insightful conversations with the Chairperson and owner of the Co-op, Mrs Dini Mardiani, as well as Mr Busono Edi Setiawan and staff member Mr Muhammad Luqmanul Hakim.The data gleaned from these interviews helped to trace the evolution and growth of stevia CM3.
In 2005, a bilateral Joint Project brought a new stevia variety, Morena, from Vietnam to Indonesia for cultivation trials.The Nukita Co-op conducted the trials in farmers' fields located in Cibodas, Bandung District.Following the project, Nukita Co-op farmers employed a simple participatory breeding approach to select the best stevia offspring.They planted the offspring obtained from generative propagation (seeds), resulting in 12 morphologically distinct lines.These 12 lines were evaluated based on their morphological and agronomic appearances for three generations, and ultimately, one line was selected: Cibodas Manis 3 (CM3).CM3 outperformed the other clones with the highest productivity and rebaudioside content, and it was also found to be more resistant to rust disease.
Farmers in the Bandung district have been growing CM3, a variety of stevia, despite it not being officially released.The primary area of development for stevia is in Cibodas, Garut, Lembang, and the surrounding regions.The Nukita cooperative provides vegetative seedlings through the Mulyasari Cibodas farmer group to aid in the development of the plant.Quality certification of the stevia seeds is ensured through BP2MB, which issues SKMB decree no.525/285/SKMB/BP2MB/2013 on December 31, 2013.BP2MB, a task force unit under the Directorate General of Plantations, is responsible for monitoring seed quality and operating the testing centre.
Nukita Cooperative began developing the stevia CM3 clone in 2010, which continued until 2019 when progress was halted by the COVID-19 pandemic.The resulting stevia products were sold as simplisia (un-processed natural ingredient), as well as 12 different types of tea (as shown in Figure 1).

Yield potential and economic value of Stevia clone CM3
The late Mr Ayip, the leader of the Mulyosari farmer group, provided information regarding the yield potential of the CM3 clone.He stated that the productivity of this clone could go up to 7 tonnes of dried leaves per hectare.This information has been supported by Sidik et al. [28].Furthermore, it has been reported that the stevia clone CM3 has a high Reb-A content, a lifespan of 6 years, and a short harvesting period of 40 to 60 days.
Based on research, it has been found that the CM3 clone boasts an exceptionally high level of productivity and quality.On the other hand, local Indonesian stevia clones, such as the BPP series clones, only yield about 2.5-3.5 tons of dry leaves per hectare per year, with stevioside content ranging from 6.2-15.1% and reb A ranging from 0.4-3.7%[18].Conversely, superior stevia clones in America produce more than 5 tons of dry leaves per hectare per year, with a total steviol glycoside (TSG) content of 15% and reb A of 9% [9].Moreover, a new variety has become available which was declared to have 21% TSG with 16% reb A [20].
It's worth noting that older stevia clones tend to have high levels of stevioside and low levels of reb A, whereas newer clones have the opposite.Reb-A is important because it provides a high level of sweetness that's similar to cane sugar and has little to no bitter aftertaste [22].Indonesian stevia development should consider using introduced clones to compete with other producing countries.These clones need to be tested for their adaptability to the Indonesian climate and soil to identify varieties that are well-suited to the region.Once selected, the introduction clones can be rapidly propagated in large numbers using tissue culture techniques [15].Before implementing the above suggestion, it is recommended to use genetic material from the local area.An economic study should be conducted to determine if cultivating stevia is feasible.Only a few studies were found to deal with CM3 economics.Sidik [26] studied the economic feasibility of stevia cultivation by the Nukita cooperative.The financial perspective of stevia cultivation, as calculated by the Nukita Cooperative, shows a high gross profit from the sale of stevia products.However, the sales turnover is still relatively small.
Based on the facts, it appears that CM3 has the potential as a high-yield stevia variety.However, it is unclear whether the uniformity and stability of CM3 seeds have been maintained.To address this concern, the performance and morphological variation of CM3 clones were observed in a farmer's field.

Performance and morphological variation of the CM3 clone
During the field study, we examined a stevia plantation owned by Jaya Giri Farmer's Group in the Lembang sub-district.The plantation used CM3 clones and organic farming methods, and the plants were 2 years old at the time of observation.Overall, the stevia plants appeared healthy and free from pests, and they did not grow too tall.This is likely due to the use of organic maintenance practices.
We conducted morphological observations following the guidelines set by the Ministry of Agriculture, specifically the PPU.Our findings revealed significant variations in morphology within the field, with at least 12 morpho-agronomic variants present in the population.These variants include branching and non-branching types, the presence or absence of hair, the intensity of hairs on the stem, leaf shape, leaf bud colour (red or green), shrub or erect type, and early or deep type.Based on these observations, breeders must select the best type (see Figure 2).These variations are likely due to the generative mode of propagation of the seedling.Overall, based on our observations, it is evident that CM3 has the potential for high-yielding stevia.However, before its release, further selection is imperative.

Conclusion
The Cibodas Manis 3 is a stevia clone developed by Nukita Cooperative, which has a high yield of up to 7 tons of dry leaves per hectare per year.Farmers in Bandung District have been cultivating this clone since and its products are sold in limited quantities as simplisia and tea-stevia-based products.Despite its potential as a high-yielding variety, this clone is not ready yet to be released due to significant morphological variations in the population.Selection efforts are required before being proposed as a high-yielding variety.Additionally, government support is needed to develop alternative sweeteners in the future.

Figure 2 .
Figure 2. Morphological variations exist in the farmer's field.