Department of Crop Improvement, CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur, India, 176062

Corresponding author Email: mr.ajitshitole@gmail.com

Article Publishing History

Introduction

Oilseed crops are the backbone of Indian agricultural economy and occupy an important position in daily diet, being a rich source of fats and vitamins. India is the second largest rapeseed-mustard growing country and accounts for 21.7% area in the world after China. Among oilseeds, rapeseed-mustard is the second most important oilseed crop of the country after groundnut and plays a significant role in Indian oil economy by contributing about 28.6% to the total oilseed production (Shekhawat et al., 2014). Rapeseed-mustard is the third important oilseed crop in the world after soybean (Glycine max) and palm (Elaeis guineensis Jacq.). The crop occupies an area of 33.58 million ha with a total annual production of 67.76 million tonnes and productivity 2018 kg/ha. In production, India ranks third after China (22.9%) and Canada (19.7%). The global production of rapeseed-mustard oil is around 12-14 million tonnes. In India, the crop occupies an area of 6.50 million ha with a total production of 8.02 million tonnes and productivity of 1262 kg/ha (Priyamedha et al., 2017).

Over the last decades, researchers have made great efforts in developing biotechnology methods to facilitate the breeding of Brassicas. Research studies indicated that the modern biotechnology will have a major impact in two areas. Firstly, it provides a new range of techniques enabling the efficient selection of favourable variants in plant breeding programmes. Secondly, it provides the opportunity to improve germplasm by increasing its diversity beyond conventional genetic limitations. Due to the relative ease of genetic transformation, Brassica oilseed crops have been amongst the first subject to study the full range of modern biotechnology methods (Abraha et al., 2008).

Conventional methods for breeding crop plants require more than six to seven years of continuous efforts to get true breeding lines after following hybridization approach, a time consuming process (Morrison and Evans, 1988). Hence, biotechnological tools including anther culture, hold a great promise in accelerating the pace of breeding programme (Guha and Maheshwari, 1964). In vitro technique of anther culture helps to achieve homozygosity very quickly (Snape, 1989). Anther culture of potential F₁ generation genotypes can be used to facilitate regeneration of stable recombinant inbreds in one to two years thereby saving time and resources for their further use directly as commercial cultivars and/or in structural and functional genomics. The object of this study was to investigate the response of different genotypes and their cross combinations for days to calli appearance.

Materials and Methods

The anther culture work was carried out in the Molecular Cytogenetics and Tissue culture Laboratory of Department of Crop Improvement, CSK HPKV, Palampur during Rabi 2010-11. The material used and methodology adopted to achieve the objectives of the investigation are given below.The material used for anther culture studies comprised of four elite genotypes and their three cross combinations (Table 1). Sufficient numbers of plants of aforementioned four genotypes and their cross combinations were raised in the pots. In order to have availability of anthers over a long period of time, plants were raised in five lots at an interval of 15 days each. For anther culture, florets from plants were clipped off when the size of bud was about 2-4 mm. The bud size was earlier established on the basis of presence of majority of the microspores at late uninuclate to early binucleate stage as studied by squashing of anthers in a drop of 1% acetocarmine. The florets of appropriate size were collected in 50 ml test tubes containing distilled water.

Table 1: List of genotypes and their cross combinations under anther culture study
Sr. No	Genotype	Parentage
1	Jayanti	Developed through irradiation from the parent variety HC-1
2	P-18	Advanced generation mutant obtained through treatment of Jayanti seeds with 0.3% EMS (Pre-Soaked)
3	P-51	Advanced generation mutant obtained through treatment of Jayanti seeds with 0.3% EMS (Pre-Soaked)
4	P(2)2	Advanced generation mutant obtained through treatment of Jayanti seeds with 90 kR dose of gamma radiations
5	Jayanti X P-18	–
6	Jayanti X P-51	–
7	Jayanti X P(2)2	–

The florets collected at aforementioned stages were treated with 70% ethanol for 10-15 seconds under aseptic conditions in a laminar air flow chamber. The florets were then surface sterilized with 0.1% HgCl₂ for 3-5 minutes with intermittent shaking followed by three washings with sterile distilled water. Florets were blot dried and opened under aseptic conditions with the help of sterile forceps and the six anthers were clipped off from each floret without damaging the anther wall. About 60 anthers were cultured in each pre-sterilized petri plate containing about 25 ml of culture medium.Two basal media viz. B₅ (Gamborg et al. 1968) and MS (Murashige and Skoog 1962) were used for callus induction. Each of these medium was supplemented with two different sucrose concentrations i.e., 3% and 4% sucrose and each of these sucrose concentrated media was also supplemented with three combinations of hormones viz. HM₁, HM₂ and HM₃ (Table 2). All the media were supplemented with 0.8% agar.The experiments on different callus induction media were replicated thrice involving different media and plant growth hormones. Anthers of all four genotypes and their crosses were plated in a replicated fashion. If there was any contamination, replating of the particular treatment was done to complete the experiment under uniform conditions. All the cultured plates were sealed with parafilm wax and kept under dark at 25 ± 1°C until calli were developed. The Days to calli appearance was calculated as follows: Days to calli appearance = Number of days taken for calli appearance from the day of culturing of anthers. Data on days to calli appearance were analyzed in Factorial Completely Randomized Design (CRD) to obtain the effect of various treatments and their interactions using statistical CPCS software.

Table 2: Different media, hormones and sucrose concentration used for calli index
Medium	Sucrose Conc.	Hormone
		Designation	Name and Concentration
B5	3%	HM1	NAA (1.0 mg/l)
B5	3%	HM2	BAP (2.0 mg/l) + NAA (2.0 mg/l)
B5	3%	HM3	2, 4-D (0.5 mg/l) + NAA (1.0 mg/l)
B5	4%	HM1	NAA (1.0 mg/l)
B5	4%	HM2	BAP (2.0 mg/l) + NAA (2.0 mg/l)
B5	4%	HM3	2, 4-D (0.5 mg/l) + NAA (1.0 mg/l)
MS	3%	HM1	NAA (1.0 mg/l)
MS	3%	HM2	BAP (2.0 mg/l) + NAA (2.0 mg/l)
MS	3%	HM3	2, 4-D (0.5 mg/l) + NAA (1.0 mg/l)
MS	4%	HM1	NAA (1.0 mg/l)
MS	4%	HM2	BAP (2.0 mg/l) + NAA (2.0 mg/l)
MS	4%	HM3	2, 4-D (0.5 mg/l) + NAA (1.0 mg/l)

Results and Discussion

Analysis of variance for days to calli appearance involving different parameters is presented in Table 3. Out of four factors, only genotypes had significant effect on days to calli appearance. Nine out of eleven interactions viz., genotypes x hormones, genotypes x media, hormones x media, genotypes x hormones x media, hormones x sucrose, genotypes x hormones x sucrose, genotypes x media x sucrose, hormones x media x sucrose and genotypes x hormones x media x sucrose showed significant effect on days to calli appearance. From the Tables 4.1, 4.2 and 4.3, it is pertinent that the effects of media, hormones and sucrose were found to be non-significant on all seven genotypes which indicated that different genotypes behaved similar in different media, hormonal combinations and sucrose concentrations for days to calli appearance. However, the genotype Jayanti x P₍₂₎₂ recorded lowest days to calli appearance on different media and sucrose concentrations. Likewise, the genotype P-51 took lowest days to calli appearance on different hormonal combinations. The effects of sucrose and hormones, hormones and media and media and sucrose on days to calli appearance are presented in Tables 5.1, 5.2 and 5.3, respectively. The results revealed that the effects of sucrose and hormones, hormones and media and media and sucrose were found to be non-significant which indicated that days to calli appearance were not affected significantly by different media, hormonal combinations and sucrose concentrations.

Table 3: ANOVA for days to calli appearance in different genotypes of Brassica carinata and their hybrids involving different media, hormones and sucrose concentration
Source of variation	df	Mean Squares	CD (5%)	CV (%)
Genotypes	6	28.23**	0.47	11.00
Hormones	2	1.38	NS
Genotypes x Hormones	12	5.17**	0.82
Media	1	0.25	NS
Genotypes x Media	6	4.67**	0.67
Hormones x Media	2	21.60**	0.44
Genotypes x Hormones x Media	12	5.04**	1.16
Sucrose	1	0.25	NS
Genotypes x Sucrose	6	0.93	NS
Hormones x Sucrose	2	63.65**	0.44
Genotypes x Hormones x Sucrose	12	5.21**	1.16
Media x Sucrose	1	1.02	NS
Genotypes x Media x Sucrose	6	12.06**	0.94
Hormones x Media x Sucrose	2	113.34**	0.62
Genotypes x Hormones x Media x Sucrose	12	7.19**	1.64
Error	168	1.02
**Significant at P ≤ 0.01

Table 4.1. Effects of media and genotypes on days to calli appearance
Media	Genotypes
	Jayanti	P(2)2	P-51	P-18	Jayanti x P(2)2	Jayanti x P-51	Jayanti X P-18	Mean	CD (P≤0.05)
MS	10.50	8.39	9.39	8.44	8.39	9.50	8.44	9.01	NS (Media)
B5	11.44	11.06	8.56	8.83	8.44	8.89	8.83	9.44
Mean	10.97	9.72	8.97	8.64	8.42	9.19	8.64
CD (P≤0.05) = 0.47 (Genotypes) CD interaction = 0.67 (Genotypes x Media)

Table 4.2: Effects of hormones and genotypes on days to calli appearance
Hormonal combination	Genotypes
	Jayanti	P(2)2	P-51	P-18	Jayanti x P(2)2	Jayanti x P-51	Jayanti x P-18	Mean	CD (P≤0.05)
HM1	13.08	9.75	10.00	10.75	9.33	10.50	10.75	10.60	NS (Hormones)
HM2	9.67	9.42	7.17	6.67	7.25	7.08	6.67	7.70
HM3	10.17	10.00	7.17	8.50	8.67	10.00	8.50	9.00
Mean	10.97	9.72	8.11	8.64	8.42	9.19	8.64
CD (P≤0.05) = 0.47 (Genotypes) CD interaction= 0.82 (Genotypes x Hormones)

Table 4.3: Effects of sucrose and genotypes on days to calli appearance
Sucrose	Genotypes
	Jayanti	P(2)2	P-51	P-18	Jayanti x P(2)2	Jayanti x P-51	Jayanti x P-18	Mean	CD (P≤0.05)
3 %	10.17	10.39	8.72	8.56	8.78	9.00	8.56	9.17	NS (Sucrose)
4 %	11.78	9.06	9.22	8.72	8.06	9.39	8.72	9.28
Mean	10.97	9.72	8.97	8.64	8.42	9.19	8.64
CD (P≤0.05) = 0.47 (Genotypes) CD interaction = NS (Genotypes x Sucrose)

Table 5.1. Effects of sucrose and hormones on days to calli appearance
Sucrose	Hormonal Combination
	HM1	HM2	HM3	Mean	CD (P≤0.05)
3 %	10.88	7.67	8.95	9.17	NS (Sucrose)
4 %	10.31	7.74	9.79	9.28
Mean	10.60	7.70	9.37
CD (P≤0.05) = NS (Hormones) CD interaction = 0.44 (Hormones x Sucrose)

Table 5.2: Effects of hormones and media on days to calli appearance
Hormonal combination	Callusing Media
	MS	B5	Mean	CD (P≤0.05)
HM1	10.05	10.95	10.50	NS (Hormones)
HM2	7.40	8.00	7.70
HM3	9.38	9.36	9.37
Mean	8.94	9.44
CD (P≤0.05) = NS (Media) CD interaction = 0.44 (Media x Hormones)

Table 5.3: Effect of media and sucrose on days to calli appearance
Media	Sucrose
	3%	4%	Mean	CD (P≤0.05)
MS	8.98	9.03	9.01	NS (Media)
B5	9.35	9.52	9.44
Mean	9.17	9.28
CD (P≤0.05) = NS (Sucrose) CD interaction = NS (Sucrose x Media)

Conclusion

The factors such as media, hormones, sucrose and their interactions viz., genotypes x sucrose and media x sucrose had non-significant effects on days to calli appearance which indicated that the genotypes behaved similar in different media, hormonal combinations and sucrose concentrations.

Acknowledgments

Authors are very much thankful to the Department of Crop Improvement, CSK Himachal Pradesh Krishi Vishvavidyalaya, Palampur for providing all the essential facilities and moral support to conduct the whole research programme and to obtain its significant findings.

References

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