In Vitro Regeneration Of Rice Biology Essay


Rice is one of the most important food crops of the world and almost all the people who lives in Asia depend on the rice for their food. Rice has great importance for biotechnology advances as a major cereal crop along with short genome sequence. However, establishment a suitable protocol of in vitro regeneration is a vital for the successful genetic transformation through biotechnology for the rice improvement. In vitro regeneration system mainly relies on totipotent explants and suitable combination of growth regulators (Hoque and Mansfield, 2004 and Joyia and Khan, 2012).

There are two major subspecies namely Japonica and Indica rice grown in diverse areas of the world. In vitro plant regeneration of Japonica rice has already been reported successfully using tissue culture system for transformation () . But there is very low regeneration ability in indica rice by employing the same protocol due to recalcitrant nature. However, indica rice varieties are unique because they have important trait for the improvement of rice and therefore there is necessary to develop a protocol for in vitro regeneration for molecular transformation.

1.1. Factors influencing the in vitro regeneration of rice

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There are many factors which affect the in vitro culture of rice includes medium composition such as basal medium (Saharan et al., 2004; Pravin et al., 2011 and Afrasiab and Jafar, 2011), hormones (Joiya et al., 2012; Chen et al. 1985), additives to the medium (Bajia and Rajam, 1995; Yang et al 1999a), carbon source (Shahnewaz and Bari, 2004 and Jain et al. 1997), gelling agents (Pravin et al., 2011 and Afrasiab and Jafar, 2011), genotype (Beena 2006; Biswas and Mandal., 2007 Khalequzzaman et al., 2005 and Zaidi et al., 2006 and Noor et al., 2011), explant material (Kishor and Reddy 1987 and Noor et al., 2011), physiological state of explant (Zhao et al., 2009) explant age (Haque and Mansfield , 2004; Amarasinghe and Yang 2005) and strategies of culture manipulation (Zheng et al. 2005).


Regeneration of rice has been investigated successfully using different explants such as mature seeds (Bano et al., 2005; Zuraida et al., 2011), coleoptile (Sahrawat & Chand, 2001), root (Hoque & Mansfield, 2004), anther (Guzman & Arias, 2000), leaf (Afrasiab & Jafar, 2011), mature embryo (Islam et al., 2005 and Noor et al., 2011) and immature embryo (Nouri-Delawar and Arzani. 2001). In in vitro studies have reported that intact leaf explants may be an excellent source in monocot crops due to presence of meristematic cells for the induction of embryogenic callus and regeneration of plants and also found that 3-4 days old seedling was good as leaf base explants (Haliloglu, 2006 and Ahmadabadi et al., 2007) and similar observation was also reported in rice (Ramesh et al., 2009). Additionally, Joyia and Khan (2012) were used green tissue from base of the stem and root portion for the in vitro rice regeneration through callus and observed that nodular calli derived from the stem segments produced shoot whereas the root derived calli only developed roots instead of shoots.

Mature embryo from dry seeds has been commonly used for callus induction and regeneration process because seeds are available throughout the year (Karthikeyan et al., 2009). Mature embryos can be used either dissected or directly. The use of mature embryos in monocotyledons is easy for the manipulation in tissue culture but the low regeneration efficiency from callus through somatic embryogenesis has been reported (Sharma et al., 2005; Chen et al., 2006 and Lee et al., 2009). It was reported that embryo excised from mature seed showed good performance for inducing callus and multiple shoot formation which could be used for transgenic studies (Azria and Bhalla, 2000). 2011). Now-a-days, a number of transformation researches have been done by using scutellum derived callus in several rice varieties ().

On the other hand, immature embryos indicate the most potential tissue of in vitro regeneration for many species. Immature embryo may be a good source due to actively dividing cells for both in vitro regeneration of recalcitrant species and rescue of interspecific hybrids. Prolific calli were produced from immature embryos after anthesis of 10 days and gave the best result for regeneration of rice (Lai and Liu, 1982). Moreover, scutellum derived callus from mature embryo of rice are also the excellent material for in vitro regeneration and even for the transformation research (Toki et al., 2006 and Shabir et al., Correspondingly, an experiment was performed successfully by Hiei and Kumari (2006) for transformation of indica rice where immature embryos were used as explants.

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But, mature embryos were performed better than immature embryo for the induction of callus and regeneration of some genotypes of rice (Noor et al., 2011).


Genotypes determine the success of in vitro rice regeneration. Khanna and Raina (1998) studied that japonica rice was more responsive to callus and regeneration compared with indica rice. Even significant variation was found in in vitro culture response of different genotypes within indica subspecies (Seraj et al., 1997, Asit et al., 2003).

According to variety, cell proliferation and regeneration in in vitro can have different genetic determinants. Beena (2006) carried an experiment with 21 rice varieties and determined that callus induction and regeneration was greatly influenced by genotypes (Beena, 2006) and this result was conformity of earlier result (Hoque and Mansfield, 2004).

Culture medium

Culture medium is one of the important factors for influencing callus induction and in vitro regeneration of rice and it differs genotype to genotype. In vitro plant regeneration of four rice genotypes, Lx297, IR64, V19 and IR64-1-1-4 was studied by using three basal media namely N6, MS and R. All varieties showed good performance for inducing callus on MS medium but the genotype, Lx297 was the best one and the highest regeneration frequency was found from callus induced on R medium when transferred to regeneration medium (Khatun et al., 2003). Similarly, Summart et al., (2008) investigated with different culture media (MS, LS, B5 and N6) on the influence of callus growth of rice variety, KDML105. Among these, N6 medium was good for obtaining average callus growth followed by LS, B5 and MS medium, respectively.

In addition, Afrasiab and Jafar (2011) conducted a study using two different media (MS and LS) and hormone combinations for in vitro regeneration of Super Basmati and IRRI-6 rice and found that MS medium enriched with different hormones was more suitable than LS medium in both varieties for callus induction but MS medium was suitable for regeneration for Super Basmati and LS for IRRI-6.

Growth regulators

Auxin and cytokinin are the two major categories of plant growth regulators (PGR) which affect in in vitro plant growth and development (Jimenez 2005). Auxin and cytokinin can be used either only or combinedly. Callus proliferated from rice explants by the addition of auxin such as 2, 4-D into a basal medium in many studies (Azria and Bhalla, 2000; Ali et al., 2004 and Toki et al., 2006 and Libin et al., 2012). Cytokinins play a key role for in vitro regeneration of rice and excellent regeneration showed by using low concentration of benzyl amino purine (BAP) into basal media (Dahleen and Bregitzer 2002 and Verma et al., 2011). In vitro regeneration does not depend only on cytokinins but also on combinations of auxins and cytokinins and it is proved in several researches (Dahleen and Bregitzer 2002, Joshi and Rao, 2009 and Joyia and Khan, 2012 Libin et al., 2012).

Besides auxin and cytokinin, other gaseous hormone like ethylene has significant effect on plant development during in vitro conditions ((Lieberman, 1979; Yang and Hoffman, 1984)) and ethylene can be regulated by manipulating the ethylene precursor and ethylene inhibitor like chemicals. 1-aminocyclopropane 1-carboxylic acid (ACC), an ethylene precursor that stimulate ethylene production by ACC oxidase (Bleecker and Kende, 2000) and aminoethoxyvinylglycine (AVG) an ethylene inhibitor which inhibit the ethylene biosynthesis by ACC synthase.


Organic supplements

Organic supplements can enhance the response of plant tissue culture in vitro. Vitamins and amino acids are commonly used as organic supplements. Vitamins are not compulsory but can improve the plant growth in culture. Among the vitamins, thiamine and myo-inositol are mostly used in tissue culture. Addition of myo-inositol into basal medium is useful to achieve the efficiency of callus induction and plant regeneration from seeds of various rice genotypes (Niroula et al., 2005)

Amino acids make available a source of reduced nitrogen. The most frequently used amino acid is glycine. Moreover, arginine, asparagine, aspartic acid, alanine, glutamic acid, glutamine and proline are also utilized. Besides, casein hydrolysate is used as a source of a mixture of amino acids and proline is a type of amino acid which provide the nitrogen to plant cells. When callus induction and regeneration medium was supplemented with casein hydrolysate and proline and it was found that callus formation was good by the addition of casein hydrolysate (Htwe et al., 2011) and regeneration efficiency was well by using proline (Khaleda and Al-Furkan, 2006).

Carbon source

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In vitro plants cells and tissues do not have autotrophic ability and thus need to apply external carbon for energy. Sucrose is low-priced, easily available, readily absorbed and comparatively stable, and is therefore the most frequently used carbon source. I is generally used at a concentration of 2- 5%. Moreover glucose, fructose, maltose, galactose, and sorbitol can also be used as carbon source and may superior to sucrose in particular conditions.

Gelling agents

Depending on the type of culture will be grown, media for plant cell culture in vitro can be prepared in either solid or liquid forms. Agar is commonly used as solidifying agent for the routine applications. Agar solidified medium was good for callus induction (Afrasiab et al., 2011). The best concentration of agar used ranges from 0.8-1.0 per cent (W/V). Gelrite, agarose, silicagel and starch copolymers are also used as substitutes of agar. In other study, gelrite (0.4%) was performed well for the highest percentage callus induction and regeneration (Shahsavari, 2010). For suspension culture, liquid medium is suitable and it is prepared without any gelling agent. For supporting the culture, filter paper bridges or glass wool can be used in liquid media cultures.

Environmental factors

In vitro tissue culture can be influenced by environmental factors like temperature, light and relative humidity etc. Generally, temperature will be depended on the type of culture and species. Temperature can be remained 25± 2oC in most of the culture. Light intensity and duration also depend on type of culture and species. Especially, only dark or light is necessary for 24 h for callus induction and 16-18 h/day and 24 h light for regeneration.

Relative humidity plays important roles in plant growth and development. The direction and rate of water flow are determined by the spatial distribution of water potentials inside and outside the vessel. Humidity inside the room should be 70-75%

1.2. Induction and maintenance of embryogenic callus

Embryogenic callus is necessary for efficient in vitro regeneration and thus transformation of any plants and embryogenic calli induction is considered the most critical step in rice because it indicate the ability of callus to form somaic embryos and therefore regenerate whole plants . Callus induction is influenced by the explants, genotypes, carbohydrate sources, hormones, basal medium and culture conditions. Particularly explants, genotype, and plant growth regulators are the important factors to achieve the embryogenic callus induction. Any tissues which have the ability of totipotency can be used for callus formation. Callus induction has been induced from several types of explants like seed, leaf, root, mature embryo, immature embryo, inflorences etc. However, scutellum derived calli of mature embryo from the seed are the best source for in vitro regeneration, and even for the making of transgenic rice (Hiei et al., 1994 and Rashid et al., 1996). In the same way, many researches have been done research on in vitro regeneration of japonica rice by using mature seed for producing scutellum induced callus ( ). On the other hand, immature embryo can be used for recalcitrant species of some plants. But, still now, there are few reports on the in vitro callus induction and regeneration of indica rice because of recalcitrance. Certainly, embryogenic callus induction is genotype-dependent. Moreover, culture media is another factor to induce calli. Employing of basal components in culture media, callus induction has been developed. Especially, the MS (Murashige and Skoog, 1962) and N6 (Chu et al., 1975) media have been extensively used in the in vitro rice culture (Ge et al., 2006). Likewise, Tariq et al, (2008) carried out an experiment through utilization of MS and N6 media containing hormone for induction of callus of four rice varieties and observed that N6 media was better than MS media. Again, Pravin et al., (2011) conducted an experiment by using different culture media (MSM, LS, L3 and MS) and reported that media had affect on two recalcitrant indica rice genotypes: Swarna and Mahsuri and found that MSM medium gave the highest embryogenic calli induction than other media.

In other study, it was found that the composition of media had a significant effect for calli production and regeneration of rice cv. Selasi and the results obtained that MSB5 medium induced more embryogenic calli than others media (NB, MS, N6). Different media, such as NB, MS, N6, MSB5 have been utilized by researchers and even though their compositions are not common, but the ratio of nitrate nitrogen to ammoniac nitrogen (NO3 −/NH4 +) is the critical parameter (Lee et al., 2002; Ge et al., 2006; Zaidi et al., 2006; Afolabi et al., 2008; Geng et al., 2008). Probably, this ratio takes part to producing somatic embryogenesis in monocots (Ge et al., 2006).

Callus induction from any tissue is augmented by the use of auxin to the culture medium. Auxin 2, 4-D is the most frequently used to induce callus from different explants of rice (Yang et al., 1999; Thadavong et al., 2002; Saharan et al., 2004; Htwe et al., 2011 Azria and Bhalla, 2000; Ali et al., 2004 and Toki et al., 2006 and Libin et al., 2012). .

It was also confirmed that callus induction was hampered in the absence of 2,4-D while Kn, NAA and BAP were used single or in combined. Besides, auxin and cytokinin can be used combined to enhance callus induction and maintenance. It was reported that the quality of initiated callus was enhanced by the addition of NAA or IAA (Ge et al., 2006). It was also investigated that organic supplements such as casein hydrolysate and proline can bed added to improve the callus induction (Khaleda and Al-Forkan, 2006).

Carbon source is one of the important factors in in vitro tissue culture systems. Although it is well known that sucrose is the excellent source of carbon for in vitro systems, but there are also some information where maltose gave better results than other carbohydrates (Chang et al., 2003; Sharma et al., 2005 and Zaidi et al., 2006). Probably, maltose may be controlled the osmotic potential of the cellular environment which allow the production of embryogenic calli (Lentini et al. 1995). Gelling agents can play the role to induce callus and regeneration in vitro. Afrasiab et al., 2011 found that agar solidified medium was good for callus formation and regeneration but in another experiment gelrite gave the better result to induce callus and regeneration (Shahsavari, 2010). Gelling agents contribute to adjust the humidity of in vitro culture conditions (Zaidi et al., 2006).

In addition to these, Phytagel® is one of the gelling agent which effective in many plant species including indica rice for the maintenance of embryogenic callus (Pons et al., 2000; Garg et al., 2002; Ma & Pulli, 2004).

Furthermore, callus induction is favoured by photoperiod. Generally, dark enhance to callus initiate and light liable to promote embryogenesis and greening of callus. Biswas et al., 2007 found that rice callus induction was better in dark than light/dark (16h light and 8 h dark) and it was confirmed by Summert et al., 2008. In contrast to this, light showed better performance for callus induction in some rice varieties (Toki et al., 2006 and Rafiq et al., 2011).

On the other hand, Summert et al., 2008 performed an experiment with two different temperatures (25±2°C or 30±2°C) on the callus induction and reported that there was no significant difference on the growth of callus but morphology of callus was totally different.

1.3. Regeneration of rice

Plant cells have the totipotent character that is responsible to regenerate a whole plant from a single cells or tissues. There are two ways by which plants can regenerate from callus such as organogenesis and somatic embryogenesis. Organogenesis is developmental method in which cell or tissues have been differentiated to induce organ. Raghavendra et al., (2009) used shoot basal portion of indica rice to induce multiple shoot directly without producing callus in a short time.

Somatic embryogenesis is a developmental route where a somatic cell or o group of somatic cells have been induced embryos. Somatic embryogenesis is a distinctive procedure in plants and it is of considerable interest for biotechnological applications like clonal propagation and genetic engineering (Quiroz-Figueroa et al. 2006, Namasivayam 2007). Specifically, somatic embryogenesis provides a valuable tool to enhance genetic improvement of crop when it is integrated with conventional breeding programs genetic manipulation (Quiroz-Figueroa et al. 2006). However, embryogenesis is more interesting technique than organogenesis, because it can be produced large number of plants in relatively short period of time because one cell produces one somatic embryo.

Regeneration of rice has been investigated using different explants such as mature seeds (Bano et al., 2005; Zuraida et al., 2011), coleoptile (Sahrawat & Chand, 2001), root (Hoque & Mansfield, 2004), anther (Guzman & Arias, 2000), leaf (Afrasiab & Jafar, 2011), mature embryo (Islam et al., 2005 and Noor et al., 2011) and immature embryo (Nouri-Delawar and Arzani, 2001).

Especially in recalcitrant crops like monocotyledonous species, immature cells and tissues are the best type of explants for plant regeneration. An efficient and reproducible plant regeneration protocols from immature embryos have been developed in several important cereals (Vasil, 1994; Repellin et al., 2001 and Nouri-Delawar and Arzani. 2001). However, the mature embryo is the best explants for in vitro regeneration from callus through somatic embryogenesis but it has low regeneration frequency (Sharma et al., 2005 and Chen et al, 2006 and Lee et al., 2009). Moreover, there is little information is available in indica than japonica rice using mature embryo because of recalcitrance (Verma et al., 2011).

In the same way, the genotype influences rice embryogenesis and thus regeneration. Although many successful reports have been described in rice and also other cereals but there is still difficulty for routine transformation of choice cultivar by reason of genotype specific response in vitro culture (Visarda and Sarma, 2004). Generally japonica was more responsive than indica cultivars in in vitro regeneration and transformation studies (Rahman et al., 2010). Even, significant variations of in vitro culture responses are observed in different genotypes within indica subspecies (Seraj et al., 1997 and Asit et al., 2003). Similar to factors like explants and genotypes, culture media also plays a role in in vitro regeneration. Biswas and Mandal (2007) performed an experiment with different media (MS, LS and N6) on the callus induction and plantlet regeneration of 10 indica and 1 japonica rice varieties and reported that callus induction and plantlet regeneration of japonica performance was better than indica in all of media.

As that of other factors plant growth regulators also plays a key role in in vitro regeneration. Exogenous auxins and cytokinins are the main plant growth regulators (PGRs) which involves to control of cell division and differentiation (Féher et al., 2003). During hormone perception and signal transduction, auxins and cytokinins interact to each other and thus plant development process (Müller and Sheen, 2008; Moubayidin et al., 2009; Jones et al., 2010). Cytokinins act as signaling molecules that influence on plant growth and development (Mok, 1994). Cytokinins play a key role for in vitro regeneration of rice and excellent regeneration obtained by using low concentration of cytokinin like benzyl amino purine (BAP) into basal media (Dahleen and Bregitzer 2002 and Verma et al., 2011). Cytokinins can play as negative regulators of root growth and development (Werner et al., 2003). Auxins are also concern to regulate on the cell cycle by controlling key genes (Blilou et al., 2002; del Pozo et al., 2002) and both hormones influence the cell cycle. In vitro regeneration does not depend only on cytokinins but also on combinations of auxins and cytokinins and it is proved in several researches (Dahleen and Bregitzer 2002, Joshi and Rao, 2009 and Joyia and Khan, 2012 Libin et al., 2012).

Moreover auxin and cytokinin, other gaseous hormone like ethylene can play important role on the plant development during in vitro conditions ((Lieberman, 1979; Yang and Hoffman, 1984)) and ethylene can be regulated by manipulating the ethylene precursor and ethylene inhibitor like chemicals into culture media.

In vitro studies have reported that ethylene can play the important role for inducing somatic embryogenesis, shoot regeneration and root formation (Chi et al., 1991; Fei et al., 2000; Khalafalla and Hattori, 2000; Lai et al., 2000 and Jha et al. 2007 and Tyagi and Dahleen, 2011). Namely, ethylene inhibitor AgNO3 has also been used effectively in many monocotyledonous species, to develop friable embryogenic callus (Vain et al., 1989; Songstad et al., 1991) and plant regeneration (Songstad et al., 1988; Vain et al., 1989). Previous result showed that shoot organogenesis and plant regeneration enhanced from cultured cells and tissues in vitro in several recalcitrant genotypes of Cruciferae family using AgNO3 and/or AVG which regulate ethylene (Pua et al., 1990 and Chi et al., 1991).

Additionally, in vitro shoot and root regeneration was good in Solanum nigrum, when silver thiosulphate was used into media as a supplement because presence of Ag+ in silver thiosulphate that inhibit the production of ethylene and thus promotes the growth of plant (Sridhar et al., 2011). On the other hand, ethylene precursor ACC has a role to induce embryoids as well as regeneration of rice (Adkins et al., 1995). Similarly,

Jha et al., 2007 conducted an experiment with six barley cultivars by adding ACC to manipulate ethylene and found the best regeneration in one cultivar 'Golden Promise' which was correlated with ethylene production. Nevertheless, it is speculated that ethylene may be regulate the ethylene receptor genes as well as ethylene signaling and therefore differentiation of cell and tissues.

Sucrose has been the most acceptable for in vitro culture of rice as carbon source and osmotic regulator. However another carbon sources, maltose and sorbotol have been used for increasing regeneration of rice. Moreover, it is proved that maltose is a better carbon source in comparison to sucrose in several studies for callus induction as well in vitro regeneration (Kumar et al., 2005 and Rafique, 2010). On the other hand, sorbitol acts as primary carbon source in tissue culture to enhance regeneration frequency of embryogenic calli (Geng et al., 2008). Callus growth stimulated and multiple shoots of regenerated plants could be obtained by supplementation of sorbitol in combination with sucrose or maltose (Cho et al., 2004 and SHAHSAVARI, 2010)

In addition to sucrose, gelling agents also control the response to tissue culture. The highest frequencies of callus induction and regeneration were obtained when 0.4% gelrite (Shahsavari, 2010). In other study, the effect of two gelling agents agar and gel rite has been assessed. Gel rite was found good to enhance callus induction and in vitro regeneration of rice varieties (Pravin et al., 2011). Although use of agar as a gelling agent to develop in vitro regeneration of rice has also been reported (Wenzhong et al., 1994; Patpanukul et al., 2004; Datta et al., 1992; Khaleda and Al- Forkan, 2006; Suprasanna et al., 2000; Lee et al., 2002) but Saharan et al., (2004) and Menses et al., (2005) have reported higher regeneration frequency in indica rice varieties with media solidified with


Though there are reports of recalcitrance among plant species to culture, this problem can be successfully overcome by manipulation of explants, culture medium or culture environment.

Culture environment such as temperature, photoperiod and light intensity are also important factor that influence the role in organogenesis and embryogenesis. Generally, the most favorable temperature for culture is 25 ± 2oC. Light also play a strong role on morphogenetic effect on plants in in vitro culture. Shoot bud formation enhanced from the callus when light intensity applied from ranged 500-1000 lux (Sompornpailin and Chutipaijit, 2012).