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Volume 1, December 2016, pages 5-12

Standardization of in vitro shoot regeneration protocol for Indian mustard,
Brassica juncea (L) using cytokinin, BAP as inducer

Geeta Dhania1*, Rana Partap Singh2

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: geetadhania@rediffmail.com | Tel: +919017567774

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Abstract

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.

Keywords

Brassica juncea (L), Tissue culture, In vitro regeneration, BAP, Cotyledon , Cytokinin

Introduction

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).
4. Discussion
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.



Conclusion

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

Acknowledgement

Authors are thankful to AAiM Edupoint, New Delhi for helping in analysis of data and compiling the manuscript.

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