Enhance The Sugarcane Productivity Using Biotechnology Approach

ENHANCE THE SUGARCANE PRODUCTIVITY USING BIOTECHNOLOGY APPROACH

Case study on Drought Tolerant, Virus Resistant and High Sugar Production Sugarcane
Presenter : Bambang Sugiharto from University of Jember, Indonesia

One of the problem that facing Indonesia is low of sugar production by sugarcane. Indonesian people are sweet lover, especially for food and beverages, that’s why in that country is high demand of the production of sugarcane to fulfill consumption of sugar. To overcome that problem Dr. Bambang was researched to enhance the sugarcane productivity using biotechnology approach which were to attained the drought tolerant sugarcane, virus resistant sugarcane, and also to produce a high sugar production sugarcane.

Seminar of Dr. Bambang Sugiharto

According to Dr. Bambang, he said that sugarcane industry mainly located in east java of Indonesia. However, sugarcane plantation and industry are located in another area of Indonesia, such as Sulawesi and Sumatra.

Indonesian sugarcane statistic 2015

The products from sugarcane industry are not only sugar, there are bagasse and molasses. The bagasse is important for producing the energy, animal feeds, and also paper. For molasses are used for cattle feed manufacturers. Sucrose is the most important product from sugarcane industry because it is used as a sweetening agent for foods and in the manufacturer like cake, candies, soft drinks, preservatives, alcohol, and so on.

Breeding is the more advanced way to improve productivity in sugarcane. The first stage of any plant breeding program is to make a cross between selected parents to develop a new breeding population that we desired. The objective is to create new clones that are better than both parents. A crucial activity for sugarcane breeders is to determine which parents to use for crossing and making new populations. Meanwhile the aims of Dr. Bambang researched is to develop and produce the drought tolerant sugarcane, virus resistant sugarcane, and also to produce a high sugar production sugarcane.

The challenges of breeding the sugarcane according to Dr. Bambang are :
1.  Sugarcane seed is very small and difficult to germinate,
2.  Poor flowering in recent sugarcane cultivar
3.  Difficulties in cross pollination among sugarcane cultivars
4. The pollen is quickly dried and can not be used for hybridization.

Thus, biotechnological approach is believed to become crucial to overcome the challenges of sugarcane breeding. Development of transgenic sugarcane may foster the development for creation of new sugarcane cultivars with various important traits such as drought tolerance, high sucrose content, resistance to diseases, high yield of ethanol and biomass for fuels.

First chapter of Dr. Bambang is to explain about the development of the drought tolerance sugarcane. He found that a drought-inducible gene, designated as SoDip22, in sugarcane leaves. He also used the gene that induces glycine betaine accumulation and developed the efficient method for genetic transformation mediated by Agrobacterium for sugarcane.This is the link that related on first chapter
https://academic.oup.com/pcp/article/43/3/350/1811894

Dr. Bambang reveals that sugarcane is a major crop cultivated in tropical fields and often suffers from drought stress. Understanding the molecular mechanism of resistance to the stress will be useful for the improvement of the yields by genetic manipulation. While searching for sugarcane proteins whose expression is controlled by drought stress, he found a 22 kDa-protein that belongs to the Asr protein family. To elucidate the physiological function, he identified the protein and the corresponding cDNA, and characterized the regulatory manner for the expression in the C4 plant, sugarcane.

He used sugarcane plants (Saccharum officinarum cv. M442–51) for development through drough stress by not watering for 8 days. He also analyze the regulation of SoDip22 by drought stress and the effect of osmotic stress. The result is amount of some proteins was decreased during drought stress. The amount of the drought-inducible protein with an apparent molecular mass of 22 kDa increased. Osmotic stress enhanced the expression of SoDip22 in the leaves.

The next development to improve the drought tolerance in sugarcane, Dr. Bambang was used the gene gycline betain (GB) for choline dehydrogenase (betA). Gorham (1995) explained that glycine betaine stabilizes the structure and activity of enzymes and proteins, and maintains integrity of membrane against damage caused by environmental stresses.

The drought-tolerance transgenic sugarcane has been developed by introduction of betA gene encoding for CDH from Rhizobium meliloti (Australian Patent Office, Patent No. 737600 – Inventor(s); Naoki Katsurada, Tsushi Hayakawa, Haruhumi Miwa). The betA gene was constructed in binary vector under the control of strong promoter CaMV35S by Ajinomoto Co., Inc., Japan and used for sugarcane transformation. The Agrobacterium mediated transformation was conducted using explant from BL sugarcane cultivars by the state-run sugarcane producer PT Perkebunan Nusantara XI Indonesia in collaboration with Ajinomoto company and University of Jember.

According to Dr. Bambang research he claimed that the transgenic sugarcane expression betA gene is a drought-tolerant sugarcane and this sugarcane should be the first drought-tolerant sugarcane developed by biotechnology approach. He successfully developed the transgenic sugarcane and it has been approved by the National Genetically Modified Product Biosafety Commission for commercial cultivation in the state-run sugarcane producer PT. Perkebunan Nusantara XI in Indonesia.

In the second part of his seminar, he explained that he developed and produced transgenic sugarcane that resistant with the virus by expression of the coat protein gene. The virus that attacked the sugarcane is SCMV sugarcane mosaic virus, this virus causes chlorosis and reducing the growth rate and also sugar productivity by 50%. So that it is crucial for enhancing the resistant sugarcane by using biotechnology approach. This is the link that correlate to second part
 https://www.ncbi.nlm.nih.gov/pubmed/30171474

Captured by Paulo Izquierdo

Dr. Bambang explained that he used method of pathogen derived resistance to develop SCMV resistant sugarcane by overexpression of viral DNA. The gene that encoded ( ScMVCp ) the coat protein of SCMV was amplified by reverse transcriptase PCR from symptomatic sugarcane leaves and used to generate transgenic sugarcane. In his research he used Agrobacterium-mediated transformation similar with drought resistance transgenic sugarcane.

He used southern blot analysis to elucidate a single hybridized DNA copy inserted into genome of transgenic sugarcane lines. The inserted genes were expressed at both RNA transcript and protein levels in transgenic sugarcane. Thus inserted genes then inserted into the binary vector pRI101-ON with two constructs, consists of full nucleotide sequence (p927) and sequence coding for N-terminally truncated protein (p702). The results from his research, he showed the highest expression in some of transgenic lines from p927 construct. From both construct he concluded that the p927 generated a highest resistance to virus compared with p702.

The transgenic sugarcane resistance to SCMV infection was successfully produced by Dr. Bambang and his teams. He adds that based on molecular analysis, the resistance is caused by introducing the full nucleotide sequence of ScMVCp gene.

In the third part of his seminar, he developed the sugarcane to produce a high yield of sugar by using the gene of sucrose phosphate synthase. Huber and huber (1996), explained that Sucrose phosphate synthase (SPS; EC 2.4.1.14) is believed to be the key enzyme controlling the flux of carbon into sucrose. Sucrose phosphate synthase (SPS) activities determine sucrose accumulation in leaf of Saccharum species. This is the link that related to third part of Dr. Bambang seminar
https://www.ncbi.nlm.nih.gov/pubmed/9327592

His research in this part, he used two cDNA clones which consists of SoSPS1 and SoSPS2 encoding SPS of sugarcane that isolated from a leaf cDNA library. Dr. Bambang revealed that for the result, he attempted to analyze the influence of sugars, like sucrose and glucose on the accumulation level of SoSPS1 and SoSPS2 with the detached leaves. However, the mRNA of SoSPS1 and SoSPS2 were rapidly decreased during the treatment with sugar and also in the control leaves supplied with water. This is due to the stress of leaf cutting. He suggest that redesigning of SPS to increase the sugar yield by genome editing.

To sum up the seminar, Dr. Bambang was successfully developed and produced the transgenic sugarcane for drought resistance and his transgenic sugarcane (GMO) was distributed to manufacturer in Indonesia legally. Subsequently, he also succeed to produce the transgenic sugarcane for resistance to SCMV virus. The last he tried to produce the high yield of sucrose by SPS activities and he found that sucrose contents was accumulated in the leaf of Saccharum species.

Writer : Salma Fuadiyah

References :
Apriasti et al. (2018). Full sequence of the coat protein gene is required for the induction of pathogen-derived resistance against sugarcane mosaic virus in transgenic sugarcane. Molecular Biology Reports. 45(6) DOI :10.1007/s11033-0184326-1
Gorham J. Betaines in higher plants – Biosynthesis and role in stress metabolism. In: Wallsgrove RM, editor. Amino Acids and their Derivatives in Higher Plants. Cambridge: Cambridge University Press; 1995. pp. 173-204. DOI: 10.1017 /CBO9780511721809.013
Huber, S.C. and Huber, J.L. (1996) Role and regulation of sucrose-phosphate synthase in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47:431-444.
Sugiharto et al. (2002). Identification and characterization of a genome encoding drought inducible protein localizing in the bundle sheath cell of sugarcane. Plant Cell Physiology 43 (3): 350-354.
Sugiharto. (2008). Biotechnology of drought-tolerant sugarcane. In : Alexandre Bosco de Oliveira (Eds.), Sugarcane Technology and Research. 201.
Sugiharto et al. (1997). Differential expression of two genes for sucrose phosphate synthase in sugarcane : Molecular cloning of the cDNA and comparative analysis of genes expression. Plant Cell Physiology, 38(8):961-965.