Geeta Dhania1*, Rana Partap Singh2
Standardization of in vitro shoot regeneration protocol for Indian mustard,
Brassica juncea (L) using cytokinin, BAP as inducer
1*Department of Environmental Sciences, Maharshi Dayanand University,Rohtak, Haryana, India.
2School of Environmental Sciences,Babasahab Bhimrao Ambedkar University, Lucknow, Uttar Pradesh, India.
* Corresponding Author Email: firstname.lastname@example.org | Tel: +919017567774
Indian mustard, Brassica juncea is of high economic importance. The crop cultivation is posed with challenges of various biotic and abiotic stresses. Since conventional breeding can be carried out in genetically compatible strains, tissue culture and genetic engineering are getting fair attention for crop improvement. Regeneration in Brassica is highly genotype dependent. We have standardized in vitro regeneration protocol for multiple shoots in Brassica juncea using BAP alone. We found 10 µM BAP in MS media induces maximum number of shoots in 100% of the cotyledonary plants. Cotyledon was found more responsive to regeneration protocol than hypocotyl. The regeneration efficiency was also influenced by age and size of explants. These findings provide new insights into the development of efficient regeneration system with high frequency multiple shoot regeneration in cruciferous plants.
Brassica juncea (L), Tissue culture, In vitro regeneration, BAP, Cotyledon , Cytokinin
Indian mustard (Brassica juncea (L) Czern & Coss 2n=36, genomic constitution = AABB) is one of the most important oilseed crop in Indian subcontinent (Srivastava et al. 2001; Shekhawat et al. 2012). It is cultivated as a winter crop in about 6 million hectares of land in rainfed areas of northern India. India is the third largest edible oil economy in the world. Among the oilseed; rapeseed-mustard (B. juncea) is contributing 32% of the oil seed production in India. The total world production of rapeseed-mustard is 32087 thousand ton from 22995 thousand hectare. India produces 4935 thousand ton from 6709 thousand hectare (Shekhawat et al. 2012). Indian mustard is an annual crop, which get matured within 90-95 days. It is generally cultivated as a rabi crop and seeded early in the spring .Seed of Brassica are small (1 kg has approx. 148,000 seeds) and have a hard seed coat. It is a predominantly a self- pollinated crop with 5-15% cross pollination.
B. juncea is a better source of lysine, methionine, cysteine, calcium, iron, manganese, selenium, phosphorus and magnesium as compared with the soybean meal. Several studies show that people with diets high in cruciferous foods have lower cancer rate (Beecher 1994). Besides, B. juncea is one of the best phytoremediators of toxic metals (like Se, Cd, Pb, Ni etc.) (Singh & Fulekar 2012).Further, the Indian mustard can also be used for gold extraction (Bali et al. 2010). The oil obtained from the mustard seeds can be used as an environment friendly, biodegradable substitute to petroleum-based fuel and for a number of industrial products such as lubricants, surfactants, surface coating (paints and printing inks), polymers and pharmaceuticals (Zaneti et al. 2013). Due to the growing world population and increasing industrialization, the demand for edible oil and biofuels is increasing; thus cultivation of oilseed crops has gained great importance (Dutta et al. 2008). Brassica juncea cultivation is beset with challenges of abiotic as well as biotic stresses. To overcome the challenges posed by the environment, workers around the globe are working to develop strategies to boost the production of the crop. Conventional methods such as, breeding although successful in some cases are not the preferred choice due to the narrow genetic base and it can be carried out only in genetically compatible crops. Due to these limitations of the conventional methods, genetic engineering is getting fair attention for crop improvement.
Abiotic stresses such as salt, drought, frost and heavy metals are multigenic traits, so it is difficult to breed the cultivars tolerant to these traits by the conventional breeding methods. Agrobacterium mediated transformation is the most popular technique for genetic transformation in plants and has been used successfully in many plant species for over a decade. A successful regeneration protocol, however, is a pre-requisite for the success of biotechnological approaches for genetic improvements in plants. Several in vitro regeneration protocols of B. juncea have been described in literature. The regeneration frequency in most of these protocols is very low, inefficient, time consuming, genotype specific and also depends on the type of explants, media composition and culture condition. In light of this perspective, the present work was undertaken to develop efficient in vitro regeneration protocols for the certain commercially important cultivars of Brassica juncea viz., Pusa Bold, T-59, RH-30, PJK, Pusa Bahar (PB), Luxmi 8812, 9304 and B9, with major focus on Pusa Bold which is compatible for in vitro selection and genetic transformation for achieving higher levels of abiotic stress tolerance in Indian mustard.
Materials and Methods
2.1. Plant material
Certified seeds of eight cultivars of Indian Mustard, Brassica juncea L. Czern & Coss viz., Pusa Bold, T-59, RH- 30, PJK, Pusa Bahar (PB), Luxmi 8812, 9304 and B9, were obtained from Pulse Research laboratory, Division of Genetics, Indian Agriculture Research Institute, New Delhi and Oil seed Section, Haryana Agriculture University, Hissar, Haryana.
2.2. Seed sterilization, germination and preparation of explants
Healthy and mature seeds of Brassica juncea were given a quick rinse in 70% ethanol and then sterilized in 0.1% mercuric chloride for 6 min. After rinsing three times in sterile distilled water, seeds were aseptically germinated on filter paper under dark. The seed coat was removed and the immature cotyledons with petiole and hypocotyl were excised from 2, 4 and 6 day old seedlings.
2.3. Culture medium and culture conditions
Cotyledon, hypocotyls explants were cultured on Murashige and Skoog’s basal medium (Murashige & Skoog 1962) containing 3% sucrose and 0.8% agar agar along with the different concentrations of BAP with or without AgNO3 . The pH of the medium was adjusted to 5.8 using 0.1N NaOH and 0.1 N HCl prior to autoclaving. The explants were allowed to regenerate in 16:8 hour light: dark period cycle with cool white fluorescent light of 80 µ M m-2 s2 at 25+2oC. For each treatment, 30 explants were cultured and each experiment was repeated at least
twice. The multiple shoots developed from the explants were counted after 3-4 weeks.
2.4. Multiple shoot regeneration
Regeneration of multiple shoots from cotyledon and hypocotyls were optimized by culturing the explants on MS medium containing different concentrations of BAP (0.1-25 µM). The effect of age of the explants on multiple shoot induction was studied by culturing the cotyledons and hypocotyl excised from the seedlings of different age (2, 4 and 6 day old).
Results and Discussions
3.1. Effect of genotype
Regeneration in tissue culture is genotype dependent. Eight commercially elite cultivars of B. juncea (Pusa Bold, 9304, RH-30, T-59, B9, PJK, Luxmi 8812, Pusa Bahar) were screened for their regeneration capability on BAP containing MS media. The frequency of shoot regeneration and number of shoot per explants varied with the cultivar. Cultivar Pusa Bold produced the maximum number of shoots per explants (11-12) in 100% of the cultures (Table 1). Since the Pusa Bold cultivar showed the best results, following experiments were carried out only on the Pusa Bold cultivar.
3.2. Effect of different explants
The 2 days old explants e.g., large cotyledon, small cotyledon, hypocotyl, cotyledon node and shoot tip responded to 10 µM BAP as induction factor. The maximum number of shoots (11-12) were regenerated in large cotyledon explants followed small cotyledon and cotyledonary node (9-10), shoot tip (8-9) and hypocotyls (6). Cotyledon explants found most responsive amongst the other explants (Table 2).
3.3. Shoot organogenesis from cotyledon explants
Cotyledon explants of Brassica juncea cv. Pusa Bold was prepared from 2-6 day old seedlings and were assessed for multiple shoot induction on medium containing different concentrations of BAP (Figure 1). On MS basal medium, cotyledon explants directly developed roots at the base in 100% of the cultures. Addition of various concentrations of BAP to the basal medium, however, induced multiple shoots at the petiolar cut region of the explants. A maximum of 11.3+0.33 shoots per ex-plant were obtained at 10 µM BAP in 100% of the culture in 2 day old cotyledon explants (Figure 1). Further increase in BAP concentration did not improve regeneration instead lesser number of shoots was developed. The length of shoots did not show any relationship with the concentration of BAP. Callus developed when subculture on to fresh medium of the same composition, did not showed regeneration. BAP is the most widely used cytokinin employed to induce multiple shoot regeneration in diverse explants of a large number of plant species including B. juncea.
3.4. Effect of different media
2-day old cotyledon explants, which were found most responsive compared to 4-6 day old explants, were cultured on MS, B5, white and SH media supplemented with different concentrations of BAP. The percentage of culture
with shoot bud differentiation and also the number of shoots per explants was maximum in MS media. A maximum number of shoots per explants were obtained at 10 µM BAP in all kind of media. Least response was observed with SH media (Figure 2).
3.5. Effect of age on donor seedling
Age of donor seedling is a critical factor for achieving high frequency of multiple shoots. Although multiple shoots were induced in explants of different age, the frequency and number of shoots decreased with increase in the age of explants. The cotyledonary explants aged 2-6 days are responsive to BAP (1 - 25 µM) to produce in vitro multiple shoots directly from the petiolar cut region (i.e., proximal end). However, the young explants aged two days responded to BAP at lower concentration (0.1 – 0.5 µM) in comparison to concentrations (1 – 25 µM) responded by the explants of 4-6 days old. Maximum % response and number of regenerated shoots were obtained in 2 days old explants cultured in 10 µM BAP. Cotyledon explants shows best response at 10 µM BAP in all age of explants. Young cotyledon explants were reported to produce optimum shoot regeneration (Figure 3).
3.6. Effect of size of cotyledon explants
The response of various portion of cotyledon explants are shown on Table 3. The cotyledonary petiole devoid of cotyledon lamina showed poor regeneration (10%) as well as significant reduction in the number of shoot per explants. The lamina alone exhibited shoot formation but the frequency of differentiation was significantly lower than that of the control cotyledon. When each cotyledon was sliced into two equal parts longitudinally and cultured on MS basal media, the regeneration response decreased (60%). These observations showed that cells most competent to differentiate shoots are located at the base of the petiole, but to express their totipotency they depend on unknown diffusible morphogenetic factor from lamina.
3.7. Effect of BAP on hypocotyl explants
The 2 day old hypocotyls explants of B. juncea cv. Pusa Bold were assessed for multiple shoot induction on medium containing different concentration of BAP (Table 4). Addition of various concentration of BAP (0.1-25 µM) to the basal medium induced multiple shoots. A maximum of 6.0 + 0.7 shoots per explants were obtained at 10 µM BAP in 20% of the cultures. At lower concentration of BAP regeneration response was high (70%), but number of shoots were 1-2 per explant. Further increase in BAP concentrations (above 10 µM) no regeneration was observed. The length of shoots did not show any relationship with the concentration of BAP.
3.8. Interaction of BAP with AgNO3
There was sharp reduction in multiple shoot formation, although the shoot length was increased with 25 and 100 µM AgNO3, when different additives were used along with BAP (Table 5).
6-Amino benzyl purine (BAP) is a cytokinin hormone of plants that is used for regeneration of shoots from explants of different tissues. Regeneration through organogenesis using various hormonal conditions has been accomplished from various tissues including cotyledons (Hachey et al. 1991;Ono et al. 1994) hypocotyls (Khehra & Mathias 1992 ; Phogat et al. 2000), peduncle (Eapen & George 1997) leaves (Radke et al. 1988), thin cell layers of epidermis and sub epidermis (Klimaszewska & Keller 1985), and protoplasts (Hu et al. 1999). Regeneration of multiple shoots in Brassica juncea has also been demonstrated in several of its strains using BAP alone or in combination of additives such as auxins (Bano et al. 2010; Kamboj et al. 2015; Trivedi & Dubey 2014).
Regeneration in Brassica is highly genotype dependent (Mollika et al. 2011; Ono et al. 1994; Zhang et al. 1998). Eight commercially elite cultivars of B. juncea (Pusa Bold, 9304, RH-30, T-59, B9, PJK, Luxmi 8812, Pusa Bahar) were screened for their regeneration capability on BAP containing MS media. The frequency of shoot regeneration and number of shoot per explants varied with the cultivar. The factors that influence the plant regeneration ability among genotype can be the altered levels of endogeneous hormones and variation in degree of differentiation in addition to the response to exogeneous hormones present in the regeneration medium. (Reddy & Reddy 1993) Cultivar Pusa Bold produced the maximum number of shoots per explants all the cultures.
Cotyledon explants have higher morphogenetic capability than hypocotyl (Fazekas et al. 1986; George & Rao 1980; Guo et al. 2005; Sharma et al. 1990). We also observed that cotyledon explants regenerated shoots at a higher frequency as compared to hypocotyls explants. Similar results were also reported earlier (Jain et al. 1988; Narasimhulu & Chopra 1988). The complete cotyledon was essential for maximum shoot regeneration potential of Brassica juncea cv. Pusa Bold. However, shoot number and percent regeneration frequency was decreased when lamina and petiole are removed. In such
explants only 1-3 shoots were observed. Thus, one may conclude that
cotyledonary lamina is crucial for shoot regeneration. However, cells most
competent to differentiate shoots are located at the base of the petiole, but
to express their totipotency they depend on unknown diffusible morphogenetic
factor from lamina (Sharma et al. 1990).
Young 2-day-old cotyledonary explants were found
more responsive to lower concentrations of BAP in MS medium as compared to the old
age explants. Young meristematic tissues are generally more responsive to in vitro culture treatment than mature
differentiated tissues (Bhojwani & Razdan 1983). Our results are not in
agreement with Sharma et al. (1990) who reported maximum number of multiple
shoots using 5-day old explants. In all the explants of different age groups,
10 µM BAP was found the best concentration to produce optimum shoot
regeneration. The regeneration efficiency was 100% i.e., all the shoots were
grown in the explants. Cotyledon explants grown on MS medium supplemented with
1 mg/L BAP induced shoot formation in 60 percent of the explants in genotypes
RH-555 whereas in RH-406 2.5 mg/L of BAP produced 65% shoot formation (Kamboj
et al. 2015). However, they did not checked concentrations higher than 5 mg/L
of BAP. We found 100% shoot
regeneration efficiency and the number of shoot formation corresponded to the
increase in the BAP concentration till 10 µM BAP, after which the number of
shoots did not increased further at higher concentrations. Similarly with
hypocotyls explants, maximum of shoots per explants were obtained at 10 µM BAP
in 20% of the cultures. At lower concentration of BAP regeneration response was
high (70%), but number of shoots were 1-2 per explant. Further increase in BAP
concentrations (above 10 µM) no regeneration was observed. The BAP when supplemented with auxins are known to
give maximum shoot regeneration at much lower concentrations (Trivedi &
Dubey 2014).The length of the shoots had no correlation with the BAP
concentrations. MS media (Murashige and Skoog 1962) was found the best medium
for shoot bud differentiations among different media tested i.e., MS, B5, white
and SH media supplemented with different concentrations of BAP. Again with each
of the media tested maximum numbers of shoots per explants were obtained at 10
µM BAP in all kind of media. SH medium was found least effective
for shoot differentiation.
Auxins and cytokinins in different combinations
have been used for induction of organogenesis in Brassica (Bano et al. 2010;
Kamboj et al. 2015; Trivedi & Dubey 2014). Success of plant regeneration
from cotyledon was variable on MS medium supplemented with different
concentrations of growth regulators. BAP alone was found to be effective in
developing efficient regeneration system giving 100 % regeneration frequency
with 11-12 multiple shoots per explant.
We have standardized a protocol for high frequency multiple shoot regeneration. We recommend 2 day old cotyledonary explants of Brassica juncea cv Pusa Bold, using 10 µM of BAP alone as induction factor in MS medium
Authors are thankful to AAiM Edupoint, New Delhi for helping in analysis of data and compiling the manuscript.
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