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TABLE OF CONTENT
Contents  Abstract  Introduction  Protocol of direct somatic embryogenesis for mass micropropagation  Advantages and comparison between direct and indirect somatic embryogenesis  Limitations and recommendations of direct somatic embryogenesis  Future prospect of direct somatic embryogenesis  Conclusion  References 16 17 12 10 8 Page 2 3 5

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1. ABSTRACT

Direct somatic embryogenesis is a process that does not involving callus stage during the development of the somatic embryos. It is one of plant tissue culture system which is potential to be manipulated for mass propagation. Previous experiment using this direct method has been published, with Saccharum officinarum, and the protocols have been welldescribed. There are various advantages from the application of this direct method that actually assist the production in order for commercialization. There are also some limitations but most of them can be counter with several steps and great strategies. Direct somatic embryogenesis is a technique that essential in manufacturing large products, with future hope on how to optimize it with other possibilities tools like disease resistant plant, recombinant protein product and new automated protocol so it is selected as powerful method to be applied for large harvesting. At the end of this reading, we should have the stand on the types of technique that suitable following the objective of this project.

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More than 30 years has passed since George Morel first proposed the application of plant tissue culture clonal propagation (Lydiane, K. et al., 2002). It was back in 1960, when George Morel first showed the potential of clonal propagation using in vitro culture technique in orchid, namely Cymbidiums using shoot tip explants (B.J. Timir et al., 2005). With several modification and improvements, Morels contribution was greatly appreciated by commercial orchid growers and they turned over to the meristem tip culture technology for mass propagation of orchids (B.J. Timir et al., 2005).

Since then, in vitro propagation or micropropagation has evolved into a competitive worldwide industry that produces 250 million plants every year (Lydiane, K. et al., 2002) and currently, micropropagation is the most commercially efficient and practically oriented plant biotechnology (A. Altman, 1998).

Figure 1: The commercially available orchids nowadays are due to George Morel s contribution in plant micropropagation

Somatic embryogenesis is a type of plant micropropagation which involves the formation of an embryo from a cell other than a gamete or a product of gamete fusion. This somatic embryogenesis is a remarkable biological phenomenon. It is an ideal system to investigate the process of differentiation in plants as well as the mechanism of totipotency in plant cell. Somatic embryogenesis has been obtained in many plant species such as Coffea Arabica, Alfalfa, Daucus carota, Brassica napus including groundnut and sugarcane, Saccharum spp. However, conversion of somatic embryos into plants remains inefficient and limits the application of somatic embryogenesis in many systems (Venkatachalam P. et al., 1999).
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Further, somatic embryogenesis can arise from two different pathways, namely direct embryogenesis and indirect embryogenesis. In direct embryogenesis, the somatic embryo develops directly from explant without intervening callus stage. This is due to the potential of the explant that already possesses the ability to form somatic embryo. Indirect embryogenesis in the other hand arises from induced callus to form the initial explant. However, regarding to the matter in finding of innovative system for producing plants by mass amount; direct somatic embryogenesis is a suitable choice with the reasons which would be explained later.

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3. PROTOCOL OF DIRECT SOMATIC EMBRYOGENESIS FOR MASS MICROPROPAGATION

Micro propagation of Somatic embryogenesis is an efficient and high volume propagation system for the production of large number of plants within a short period (Parrott, W. A., Merkel, S. A. and Williams, E. G, 1991). Plant regeneration through somatic embryogenesis has been reported in sugarcane using young leaf rolls and immature inflorescence (Ho, W. J. and Vasil, I. K, 1983)

Embryogenic callus was induced in the presence of 2,4- dichlorophenoxyacetic acid (2,4-D) or picloram and regeneration was obtained by reducing the concentration of the auxin or deleting it from the medium (Fitch, M. M. M. and Moore, P. H, 1990) or by media supplementation with thidiazuron (Gallo-Meagher, M., English, R. G. and Abouzid, A, 2000). Below is the protocol of using immature inflorescence segment in direct somatic embryogenesis method.

3.1 Plant material and establishment of cultures

Saccharum officinarum, coC-671 was selected. Fresh plant material of immature inflorescence segment was collected form 10-month-old field-grown plants. The material was washed several times with tap water with few drops of liquid soap. It is better washed with sterile water for 60 minutes to remove major bacteria. After it, outer old leaf-base coverings were removed carefully without damaging the inner young and delicate tissue flowed by the immersion of portion of inflorescence in absolute alcohol for 5 minutes to further the surface sterilization. The outer sheaths of the material was removed and cut the innermost inflorescence segments into 3-6 mm long pieces and inoculated on Murashige and Skoog (MS) medium (Murashige, T. and Skoog, F, 1962) supplemented with different plant growth regulators and additives. (shown in table 1). pH of the medium was adjusted to 5.8 and autoclaved at 15 1b for 15 minutes. The segments were cultured about 3-6 weeks initially in the dark period and there after all the cultures were incubated in the culture room at 26 1C under cool, additional whitle fluorescent light (1000lux) for 12 h/day, with relative humidity of 70-80%.
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Presence of somatic embryos on the cultured explants after four-week culture period was the initial succeed of protocol with the mass number of regenerated plants per explants after eight-week culture period. For each treatment, 70 80 explants were employed and the experiments were repeated at least thrice and the data were statistically analysed.

3.2 Histology

Different development stages of somatic embryos were monitored by observing the cultures under stereo-microscope. To study the different developmental stages of the explants for direct somatic embryogenesis, thin sections were cut at 15 mm thickness using disposable blade, carried out in a cryotome (Lica). Good and intact sections were mounted in drops of aqueous safranine (2%) and observed under the microscope. The photomicrography was carried out using Zeiss Axioplan compound microscope.

3.3 Acclimatization of regenerated plants

Well-developed somatic embryos were cultured on MS basal media for germination. Agar was washed-off carefully under running tap water and germinated embryos with good root and shoot system (emblings) were allowed to grow in small plastic pots containing autoclaved soil, covered with polythene sheets and maintained under 25C temperature and 70% relative humidity in the greenhouse or Reactor of Automatized Temporary Immersion (Alvard et al., 1993) (shown in Figure 1)

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4. ADVANTAGES AND COMPARISON BETWEEN DIRECT AND INDIRECT SOMATIC EMBRYOGENESIS

4.1 Advantages direct somatic embryogenesis

Direct somatic embryogenesis has many advantages in plant tissue culture such as first, genetically identical plants can be produced. As somatic embryogenesis is initiated directly from explants, the clones are identical. It is also considered safe since there was no risk of producing plants that are genetically different from parent plant. Besides that, there are few benefits in terms of environmental control, for instance, a large number of individual cells can be handled in small space, and the growth of plants can be controlled variably, and plants can be grown in aseptic condition. Direct somatic embryogenesis also enables the genetic improvement in plants, where the single cell can be modified. Plant tolerance to abnormal temperature, herbicides, fungal toxins, high salt level and disease resistance can be improved by exposing cell cultures to a selective agent. As the direct somatic embryogenesis does not intervene with callus stage, the time period for embryogenesis is shortened. Direct somatic embryogenesis also provides an efficient system of plant regeneration (Hall. R.D, 1999) because in indirect somatic embryogenesis, the friable embryogenic callus should produce mature somatic embryos before they germinate. But, maturation occurs at very low frequencies and friable embryogenic callus cannot be used to efficient plant multiplication. Another advantage is that mature somatic embryos are ideal explants since they can produce millions of propagules. Furthermore, somatic embryogenesis has developmental programs to grow into complete plant without mechanical isolation and separate shooting and rooting steps. Hence, labor costs can be reduced intensively. Direct somatic embryogenesis also reduces somaclonal variation because the number cell generations in the disorganized growth phase in minimized. The generation of plants with abnormal, ill adapted plants, are particularly unwelcomed because the consequences may be worse later when the plant have long life cycle.

4.2 Comparison between direct and indirect somatic embryogenesis

Direct Embryo is produced directly

Aspect Development

Indirect Callus is produced

8

from

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TYPE THE DOCUMENT TITLE from a group of cells(explants) without intervening callus stage 2 distinct stages: a) embryo initiation b) embryo production Stages explants,

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and

embryo

is

produced from callus tissue or callus cell suspension Callus production followed by embryo initiation

Plants produced are identical, Genetic variation no variation

Somaclonal variation due to dedifferentiation of tissues during callus stage

Less risky as plants produced Risk identical to parent plant, no somaclonal variation Shorter time period as no Time period callus stage.

Risk present as somaclonal variation may cause

abnormal, ill-adapted plants Longer time required as

explants has to pass callus and then embryo stage

Not favoured as somaclonal Production variation does not take place

of

genetically Favored dedifferentiation of

since cells

modified plants

occur at callus stage. Cella at callus stage can be induced by mutation to

produce genetically different cells. Efficient since matured Efficiency Depends to callus culture, and from callus. regeneration friable success

embryo is used for explants regeneration

embryogenic

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5. LIMITATIONS AND RECOMMENDATIONS OF DIRECT SOMATIC EMBRYOGENESIS

5.1 Limitations

Very stringent environmental conditions are required for the success of a direct somaclonal embryogenesis. These include a high concentration of auxin that is often required for embryo induction. But on the contrary, for further development of the embryos, auxin concentration must be lowered or completely eliminated from the medium. High temperatures are also needed for the development of an embryo (Islam T., n.d.). Optimization of the culture medium is very difficult and an elaborate and labour-intensive experimental set-up is needed. (Strosse H.,1995) .Another limitation of this route of embryogenesis is that the response of embryogenesis is tissue specific and confined to a few species only (Islam T., n.d.). For example, in a study done on peanuts by Venkatachalam P. et al (1999), it showed that the present findings on auxins (NAA)/Benzylaminopurine (BAP)-stimulated embryo induction are in contrast to all previous results in peanuts but differ from previous findings with legumes in general. Auxin 2,4-D and NAA were found to be effective in inducing somatic embryos in peanut. Cytokinin-stimulated direct somatic embryo induction from non-embryonic tissue was also effective. Therefore, it can be seen that a generalized protocol for somatic embryogenesis in grain legumes are difficult. The growth regulator requirements are species-and tissuespecific. Barriers can also occur anywhere between the inductions of plantlets to the production of plantlets that are capable of surviving transfer from in vitro conditions to ex vitro conditions. These barriers may include a low production of embryos, malformation of the embryos, incomplete embryo maturations, unbreakable embryo dormancy or low plantlet vigour. All these may create unwanted genetic variation (somaclonal variation) (Islam T., n.d). Usually, successful genetic transformation has used embryonic callus/cell cultures as the target tissue in crop plants, including sugarcane. However, this callus system causes a major limitation as repeated subcultures need to be done to select embryogenic callus portions among highly proliferating non-embryonic tissue. This process increases the chances of somaclonal variation (Desai N.S. et al.,2004).Unpublished results also revealed that somaclonal variation increases as culture time is increased (Cte and colleagues, unpublished data).

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The need for surface sterilization during culture establishments using toxic surface sterilants is one of the limitations that arise. Besides that, there are phenolic secretions during culture initiation too. To overcome these problems, the use of inflorescence culture system in direct somatic embryogenesis is recommended. It does not need surface sterilization and there is no phenolic secretion. Hence, the method is free from the problem of phenolics. (Desai N.S. et al.,2004)

5.2 Overcoming the limitations

Overcoming somaclonal variation could be achieved by finding molecular markers of somaclonal variartion. These kinds of markers would help scientists understand how somaclonal variation is brought about and to help identify the factors that influence its development. Data using flow cytometry would also be a great help to allow scientists to better understand how somatic embryogenesis techniques generate somaclonal variants (Roux et al, in press). In the research done by Snyman S.J. et al (2006), reducing the exposure to growth hormones can also decrease somaclonal variation. To optimize the culture medium for direct somatic embryogenesis, a few suggestions were made by Strosse H. (1995). The first is to optimize the starting material such as the size, and osmotic state of the explants. The osmotic condition of the culture medium and the concentration of the auxins that are responsible for the unorganized growth of the embryonic cells should also be changed. The culture media should be added with embryogenesisinducing compounds such as amino acids and polyamines. Physiological conditions of the culture and explants should also be changed to suit the different species of plant such as pH, temperature, and humidity. Nurse or feeder cultures can also be applied. Feeder cultures are cultures that secrete growth factors and are normally prepared from splenocytes, macrophages, thymocytes or fibroblasts. They are used as a substitute for conditioned medium. Last but not least, a procedure that can obtain embryogenic callus based on the successive use of two culture mediums could be set up.

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6. FUTURE PROSPECT OF DIRECT SOMATIC EMBRYOGENESIS

6.1 Sugarcane biotechnology: the challenges and opportunities

Biotechnology offer opportunities for sugarcane crop improvement. Commercial sugarcane mainly the interspecific hybrids of S.officinarum (Bakker, 1999) will have great advantage and benefit as a results of biotechnological improvement due to its complex polyploid aneuploid genome, narrow genetic base, poor fertility, and the long duration (12 15 years) required to breed elite cultivars. There is increasing pressure worldwide to enhance the productivity of sugarcane cultivation in order to sustain profitable sugar industries (Hanlon et al., 2000). Advancement in biotechnology will offer opportunities to address issues related to development of novel, high yielding cultivars. (Briggs and Koziel, 1998; Ellis et al., 2000).

6.2 Resistance to disease and pests

Sugarcane is susceptible to a host of viral, bacterial and there is also seven recognized sugarcane disease of unknown aetiology (Rott et al., 2000). The approaches of developing resistance against the viral disease is by applying pathogen-derived resistance (PDR) genes which is the genetic elements originated from and conferring resistant against pathogen itself (Abel et al., 1986).The PDR genes have been engineered into plants to develop virus resistance include full length, truncated or mutated virus coat-protein. Many elite sugarcane clone are susceptible to different fungal and bacterial disease therefore limit their commercial exploitation. Little knowledge is known about the molecular basis of pathogenesis of variety fungal and bacterial diseases of sugarcane. Recently a number of molecular strategies to confer resistance against fungal and bacterial pathogen are tested in many crop species (Hancock and Lehrer, 1998; Mourgues et al., 1998; Bent and Yu, 1999; Cao et al., 1999; Osusky et al., 2000). These include the use of antimicrobial proteins (Harrison et al., 1996), genes that inactivate pathogenicity (Zhang et al., 1999), natural disease resistance genes (Staskawicz et al., 1995), phytoalexins (Glazebrook et al., 1997), and antimicrobial peptides (Hancock and Lehrer, 1998; Osusky et al., 2000). Many of these genes have been successfully used to control plant fungal and bacterial diseases (Terras et al.,
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1995; Krishnamurthy et al., 2001; Chakrabarti et al., 2003). Sugarcane pests are another major cause of economic loss in all the cane-growing countries, including Australia. Presently sugarcane pests are controlled by integrated pest management such as biological control. Increasing pest resistance by introduction of novel insectidal genes by transgenic approach will be another technology to help in maximizing and sustaining crop productivity. (Allsopp and Manners, 1997; Legaspi and Mirkov, 2000; Falco and Silva-Filho, 2003). Transgenic sugarcane engineered with the Nicotiana alata proteinase inhibitor gene (Atkinson et al., 1993) exhibited marked antibiosis to canegrub (Allsopp et al., 2000; Nutt et al., 2001). The transgene can be alternative source of resistance that can enhance IPM strategies to provide opportunities to pyramid natural and transgenic pest resistance genes into breeding and commercial germplasm.

6.3 Sugarcane as a biofactory: Production of high-value alternative products

Alternative products to sucrose can give not only higher value product but also make sugar industries more sustainable and competitive. Sugarcane has all features that are required for a natural biofactory: it grows rapidly, has efficient carbon fixation and possessed a well-developed storage system. Development of plant-based expression system has produced over 100 recombinant proteins successfully produced in plant species. These expressed proteins include a wide range of products from viral proteins, vaccines, antimicrobial peptides, antibodies, pharmaceuticals, and industrial compounds (Twyman et al., 2003). Sugarcane also prove to be a model system for production of poly-3-hydroxybutyrate (PHB). Sugarcane as a biofactory for industrial compounds also be used in production of p-hydroxybenzoic acid (pHBA) from transgenic sugarcane expressing the bacterial enzymes chorismate pyruvate-lyase (CPL) and 4-hydroxycinnamoyl-CoA hydratase/lyase. Although the opportunities to develop sugarcane as a biofactory are expanding there are several obstacles that remain to be solved. The issue is like control transgene expression and biological activity of its products. Recent research prove consistent and impressive levels of transgene expression can be achieved by expressing transgene within plastids (Devine and Daniell, 2004; Maliga, 2004). Chloroplast
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transformation has been used to express protein-based polymers (PBP) at 100-fold higher efficiency than nuclear transformation (Guda et al., 2000). In Australia, efforts are now under way to develop a plastid transformation system for sugarcane. We believe that the technological innovations happening in plant transgenic research will undoubtedly provide all the necessary tools to develop sugarcane as a commercially viable biological platform for high-value products in the near future.

6.4 Structural and Functional Genomics

The evolution of structural and functional genomics will make major impact on future sugarcane crop improvement programs. These advances include the development of highly efficient DNA sequencing technique, innovative approaches to the identification of SNP s and genome mapping, a variety of powerful DNA microarray technologies for the analysis of gene expression, RNAi (RNA interface) technology and development in data mining tools to organize and identify the complex data produced from these technologies. This advancement in technology now offers an unprecedented analytical power in studying agriculturally biological processes. The most challenging phase of sugarcane genomic research is deciphering gene function. A large collection of sugarcane ESTs, nearly 260 000, generated mainly by Brazilian researchers (Vettore et al., 2003) are now available in public domain and will stay as a highly valuable resource for genomics research for a long time. A powerful method by using cDNA microarray technology to identify regulatory genes that related with a particular process in sugarcane. cDNA substractive hybridization and cDNA macroarray strategy is used to identify sugarcane ESTs differentially expressed in immature and maturing intermodal tissue which prove that a combination of cDNA substraction with macroarray screening is an good way to analyze developmentally regulated gene in sugarcane. Functional genomics will soon emerge as one of the most powerful approaches to solve the issue related to genetic improvement of sugarcane along with the rapid advances located in microbial, plant, and animal systems.

6.5 Simple and reproducible protocol for direct somatic embryogenesis from cultured immature inflorescence segments of sugarcane
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Direct somatic embryogenesis offers several advantages in crop improvement, as cost effective and large-scale clonal propagation is possible by operating with bioreactor that will lead to automation of somatic seed production and the development of artificial seeds. The system also provide a new source for use in genetic transformation. The immature inflorescences of graminaceous plants have become excellent source of young meristematic tissue for the induction of direct somatic embryogenesis. In the process of direct somatic embryogenesis, the part of sugarcane that is used is young leaf rolls and the culture system has been employed for genetic transformation. The immature inflorescence of sugarcane consists of main rachis and many sub-rachii and is delicate and fragile, light to dark pink in colour. Advantageous of using immature inflorescence explant as they are discarded and therefore sacrificing the plant for culture initiation is unnecessary. The inflorescence material is readily available and free from any disease. Therefore surface sterilization with toxic surface sterilants is not needed. There is also no phenolic secretion during culture initiation. Callus-free and regeneration pattern through induction of direct somatic embryogenesis since callus culture is associated with variety of problems of embryo formation, maturity and plantlet regeneration. The callus culture system is of genomic instability and regeneration problem. The induction of direct somatic embryogenesis will produce a large number of plants in a short time. The induction of direct somatic embryogenesis using inflorescence segments can be useful inrapid propagation of elite sugarcane varieties. A system will allow transformation process such as Agrobacterium or particle bombardment to be more successful by producing transformed plants in a short time that will prevent somaclonal variation.

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TYPE THE DOCUMENT TITLE 7 CONCLUSION

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After discussing the information above, we can conclude that direct somatic embryogenesis is a suitable innovative system that can be utilized in this real world, delivers great impact especially in economic field. This is one way to help the small farmers to get their large profits and a best way to expand the companys production across the countries. It is a dream that every companies plant-based product are hoped on to have less time, cost and risk but high yield to commercialize their product. Hence, it is important for this system to be installed in our company with better next coming stocks production.

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[Pick the date]

29. Zhang, L., Xu, J. & Birch, R. G. (1999). Engineered detoxification confers resistance against a pathogenic bacterium. Nature Biotechnol, 17:1021 1024.

30. Zimmerman J.L. (1993). Somatic Embryogenesis: A model for early development in higher plants. American Society of Plant Biologists. Retrieved April 12, 2010, from http://www.jstor.org/pss/3869792

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