Analysis of Trait Variation and Correlations in Hybrid Rice Seed Production Using CMS Breeding Techniques

 Riya Pal1* and Chand Kumar Santra2

1Department of Botany, The University of Burdwan, Burdwan, West Bengal, India.

2Department. of Agriculture (Research Wing), Government of West Bengal, Rice Research Station, Chinsurah, Hooghly, West Bengal, India

Corresponding Author E-mail: riya.pal20@gmail.com

Article Publishing History

Received: 16 Sep 2024
Accepted: 09 Dec 2024
Published Online: 17 Dec 2024

Review Details

Plagiarism Check: Yes
Reviewed by: Dr. Manohar Saryam
Second Review by: Dr. Abdal Ahmed
Final Approval by: Dr. Torit Baran Bagchi

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Abstract:

Imperfect panicle emergence in Cytoplasmic Male Sterile (CMS) lines is a hindrance for hybrid rice seed (F1) production. In CMS lines 10-15% of spikelets remaining enclosed within the flag leaf. This limits their availability for outcrossing, thereby reducing the efficiency of hybrid rice seed production. To mitigate this issue, the application of gibberellic acid (GA3) is a common practice. However, the high cost of GA3 poses a significant barrier for middle-class farmers involved in hybrid rice seed production. This study aims to identify low-cost chemical alternatives to GA3. We evaluated ten different treatments, including a control, on CMS (A) line rice plants. Our findings indicate that penicillin is the most effective treatment, offering high yield at a reduced cost. According to the yield component, treatment-2 (penicillin) reflected grain yield 71.26 g plant -1 which is highest among all the treatments.  Potassium dichromate also performed well in promoting panicle emergence. Characters showing positive and significant correlations among different traits are expected to improve yield and can be selected for further advancement in hybrid rice seed production.

Keywords:

Chemical alternatives; Correlation; CMS; F1; Panicle emergence; Penicillin

Copy the following to cite this article:

Pal R, Santra C. K. Analysis of Trait Variation and Correlations in Hybrid Rice Seed Production Using CMS Breeding Techniques. Curr Agri Res 2024; 12(3).

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Pal R, Santra C. K. Analysis of Trait Variation and Correlations in Hybrid Rice Seed Production Using CMS Breeding Techniques. Curr Agri Res 2024; 12(3). Available from: https://bit.ly/3ZC6skJ


Introduction

Essentially, hybridization is the most potential breeding method for improvement of crops. Hybrids are effective only when such product shows heterosis in yield over traditional popular varieties to become commercially successful. The three-line hybrid rice system involves three distinct parental lines: The A line (Cytoplasmic Male Sterile, CMS line), the B line (Maintainer line), and the R line (Restorer line). Each line plays a critical role in the production of hybrid rice, contributing to sterility, maintenance, and fertility restoration, respectively. Hybrid rice is a crop of cross between two genetically dissimilar parents. The foundational work on the development and utilization of hybrid rice technology in India began in the 1970s. A significant milestone was achieved in December 1989, when the Indian Council of Agricultural Research (ICAR), New Delhi, launched a time-bound, goal-oriented network project, prioritizing hybrid rice in the national agricultural agenda. Improving the efficiency of hybrid seed production requires enhancing the outcrossing capacity of Cytoplasmic Male Sterile (CMS) lines to increase hybrid seed yield.¹

Gibberellic acid (GA₃) significantly enhances stigma exsertion rates, extends the duration of floret opening, and improves panicle exsertion from the flag leaf sheath. These effects contribute to improved outcrossing efficiency, making GA₃ a valuable growth regulator in hybrid rice seed production.

Additionally, GA₃ influences plant height, often resulting in taller plants with improved productivity, making it a crucial growth regulator in hybrid rice cultivation.2-5Gibberellic acid (GA₃) is an effective but costly chemical, posing a challenge for adoption by middle-class farmers. Growth regulators such as GA₃, indole-3-acetic acid (IAA), and naphthalene acetic acid (NAA) have been shown to enhance floral traits and improve outcrossing rates in various Cytoplasmic Male Sterile (CMS) rice lines, thereby boosting hybrid rice seed production.⁶ The application of GA₃ led to the highest plant height and panicle length, followed by the combination of Brassinolides and GA₃, indicating their effectiveness in enhancing panicle exsertion. GA₃ application alone resulted in significantly higher seed yield, followed by the Brassinolides + GA₃ combination, and then Brassinolides alone across both CMS lines in both the  seasons. Hybrid rice seed yield reached up to 276 g in treated plants compared to 73 g in the control, with the increase over control being higher in CRMS 32A (80%) than in IR 62829A (54%). Additionally, seed set percentage showed a significant positive correlation with seed yield.7 The treatment combination T26 (GA₃ 45 kg + Urea 10 g + Boric Acid 2 g + ZnSO₄ + K₂PO₄ 2 g) yielded optimal results, suggesting it as an effective approach to enhance hybrid rice seed production. This treatment combination has potential as a substitute for GA₃ in India and other hybrid rice-producing countries.8 The highest seed yield in CMS lines could be achieved with the application of GA₃ at 30 g ha-1 combined with Nutragold, highlighting its effectiveness in promoting hybrid rice seed production.For improving yield, correlation is instrumental in identifying suitable selection criteria and  yield-based direct selection may be beneficial.  So, it is essential to know the relationship between morpho-agronomic characteristics and seed production for a successful selection process.10

The aims and objectives of this study is to explore low cost alternatives other than GA3 to combat the unexsertion of panicle of CMS (A) line and enhance hybrid rice seed production.

Materials and Methods

IR58025A (CMS line) and KMR3 (R-line) were procured from the Rice Research Station, Chinsurah, Hooghly, and grown in the research plot at the Crop Research Farm under the Department of Botany, University of Burdwan, Tarabag, Bardhaman, West Bengal during the boro season.The experiment was conducted using a randomized block design (RBD) with three replications during the boro season of 2013-14.In this study, the R line and A line were transplanted at a ratio of 2:6.Treatments were applied as foliar spray on A line plants for F1 hybrid rice seed production. Treatments and doses of chemicals are exhibited in (Table 1). The characters studied were  (i) plant height (cm) (ii) no. of tiller plant-1(iii) total panicle length plant-1(cm) (iv)  panicle exertion length (cm) (v) total no. of grain panicle-1(vi)  no. of fertile grain panicle-1(vii) grain yield plant-1and (viii) 1000 grain weight (g).All the characters were also taken into account for calculating its analyses of variance (F value), its critical difference (CD) and coefficient of variation (CV) values. Similarly, other components of variances like genotypic variance (δ2g), phenotypic variance (δ2p),environmental variance (δ2e), genotypic and phenotypic coefficient of variations i.e. GCV and PCV including heritability (h2) were also estimated. For analysing correlation values (r), some metrical characters viz. plant height (cm), total panicle length plant-1 (cm), panicle exsertion length plant-1(cm.), grains panicle-1 (no.), fertile grain panicle-1 (no.), 1000 grain weight (g) were selected.

By the assessment of value of variance i.e. ‘F’ value was noted whether it is significant or not. When the ‘F’ value was significant in order to compare the error mean square (EMS) and the degree of freedom (df) we calculated the critical difference (CD) by the following formula:

Where, SE is standard error of the difference of the treatment means to be compared, and  is equal to:

With MSe as error mean sum of square and r as the number of replications, and ‘t’ is the tabulated value at 5% or 1% level of significance for the degree of freedom of error mean square.Thus,

The coefficient of variation (CV) was also calculated by the following formula:

Accordingly, the components of variances were also calculated by the following equation:

Thus, the genotypic variance being δ2g and the environmental variance as δ2e, the phenotypic variance, i.e.

The components viz. δ2p, δ2g and δ2e were used for estimation of other useful statistics such as phenotypic coefficient of variation (PCV), genotypic coefficient of variation (GCV), as followed by Singh and Chaudhary.11 The broad sense heritability (h2) was also calculated in all metrical character by the formula as :

Pearson’s correlation coefficient is given by:…x)

rxy = correlation coefficient between x and y (Pearson’s correlation coefficient)

xi = the values of x within a sample

yi = the values of y within a sample

x ̅= the average of the values of x within a sample

y ̅= the average of the values of y within a sample

Table 1: Treatments and doses of chemicals

Treatments

Chemicals applied

Doses (ppm)

T1

Control

T2

Penicillin

400

T3

Sulfonamide

100

T4

Gentamycin

100

T5

GA3

60

T6

Nickel chloride

100

T7

Potassium dichromate

100

T8

Lead acetate

100

T9

Copper sulphate

100

T10

Ammonium molybdate

100

Results

Mean values of various metrical characters are exhibited in (Table 2).

Table 2:  Data showing mean values of various metrical characters at a glance                  

Season

Treatment

Plant height (cm)

No. of tiller plant-1

Total panicle length(cm)  plant-1

Panicle exsertion length (cm)

Total no. of grain panicle-1

No. of fertile grain  panicle-1

Grain yield plant-1 (g)

1000 grain weight (g)

 

(Boro-2013-14)

 

 

T1

79.66

10.16

22.81

19.41

190.05

75.55

68.03

18.88

T2

83.78

11.53

25.75

22.61

211.95

81.81

71.26

21.20

T3

79.73

10.74

23.76

20.06

210.63

78.95

69.40

20.35

T4

79.23

10.87

23.91

19.81

209.56

79.04

69.26

20.20

T5

82.61

11.32

24.76

21.46

210.58

80.70

70.60

20.78

T6

82.76

11.10

24.50

21.18

208.26

79.85

70.46

20.45

T7

81.66

10.83

24.68

21.32

208.25

79.76

69.66

20.28

T8

80.95

10.71

24.51

20.45

195.76

78.47

60.40

20.25

T9

79.03

10.72

22.96

18.10

193.63

76.95

54.90

18.71

T10

76.46

10.17

23.25

19.93

190.15

75.56

67.78

18.81

Table 3: Combined ANOVA for various metrical characters

Click here to view Table

Table 3: Continued…

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Table 4: Correlation values (r) at a glance

Sl. No.

Characters

Correlation values (r)

Remarks

1.

Plant height (cm) vs.

total panicle length plant-1(cm)

0.8395

Positively correlated and significant at 1%

2.

Total panicle length (cm) plant-1(cm) vs. fertile grains  panicle-1 (no.)

0.6533

Positively correlated and and significant at 1%

3.

Total grains panicle-1 (no.) vs. grain yield plant-1 (no.)

0.6108

Positively correlated and and significant at 1%

4.

Total panicle length plant-1(cm) vs. panicle exsertion length plant-1

0.9277

Positively correlated

and significant at 1%

5.

Fertile grains  panicle-1 (no.) vs.1000 Grains weight (g.)

0.9570

Positively correlated and significant at 1%

Correlation values (r) are graphically represented in Fig.1 (a-f).

Figure 1: Graphical representation of correlation values (r):(a) Plant height (cm) vs. total panicle length plant-1(cm), (b) fertile grains panicle-1 (no.) vs. total panicle length plant-1(cm)

Click here to view Figure

Discussion

 Various metrical characters under different treatments were studied carefully till harvesting. In this experiment it is recorded (Table 2) that plant height was highest (83.78 cm) in treatment-2 (penicillin) and lowest (76.46 cm) in treatment 10 (ammonium molybdate). Highest number of tiller (11.53) was found in case of treatment-2 (penicillin) and lowest was (10.16) in case of treatment-1 (control). An increased number of effective tillers plant-1 is closely associated with higher seed yield  plant-1, which subsequently contributes to enhanced overall productivity. This trait plays a critical role in determining yield potential in hybrid rice production. The exogenous application of NAA at 90 ml ha-1 during the panicle initiation and tillering stages significantly enhanced yield in coarse rice (IR-6), resulting in greater net returns. Thus NAA application increases yield potential and profitability in rice cultivation.12

Similarly, total panicle length plant-1was also highest (25.75cm) in case of penicillin treatment. GA3-T5 also showed good result in this regard.  Among the treatments, penicillin (treatment-2) exhibited the highest panicle exsertion length (22.61 cm), while the control (treatment-1) showed the lowest (19.41 cm). GA3 treatment resulted in an exserted panicle length of 21.46 cm, and potassium dichromate recorded a panicle exsertion of 21.32 cm. GA3-T5 and potassium dichromate-T7 exhibited good results in case of panicle emergence.  The parental lines of hybrid rice KRH 2 (CMS line IR-58025A and restorer line KMR 3) treated with five GA₃ doses and a water control in a randomized block design with three replications.13 Across both seasons, GA₃ application at 200 ppm was most effective in enhancing spikelet fertility in the CMS line, positively impacting seed yield. The highest seed yield (2.18 t ha-1) was observed with 200 ppm GA₃ during the kharif season, while a slightly lower yet significant yield (2.15 t ha-1) was achieved with 100 ppm GA₃ during the boro season. This suggests that GA₃ concentration may be optimized seasonally for maximizing hybrid rice seed production. Grain yield in case of penicillin treatment was 71.26 g plant -1 which was the highest among all the treatments. No. of fertile grain was also highest (81.81g) in case of penicillin treatment and it was lowest (54.90 g) in case of treatment 9 (copper sulphate). Potassium has been shown to promote panicle development and enhance rice yield, particularly at higher application rates.14 Conversely, higher concentrations of copper sulphate (100-250 mg kg-1) have been found to reduce growth, dry matter production, and nutrient content in greengram.15Lead compound also showed decline in productivity.16The analysis of variance revealed that F-values for all metrical characters were significant at the 0.01 probability level, with treatment as the source of variation. Heritability (h²) values ranged from 0.97 to 0.99, with the highest value (0.99) recorded for traits such as the number of tillers plant-1, total grains panicle-1, fertile grains panicle-1, grain yield  plant-1 (g), and 1000-grain weight (g). High heritability, coupled with substantial genetic advance and significant genotypic and phenotypic coefficients of variation, suggests strong potential for the genetic improvement of this traits.17

The component of variances both genotypic and phenotypic gave the significant data. Higher values were found  in case of δ2g  for the metrical characters like number of grain panicle-1 (85.21), number of fertile grains panicle-1 (4.40) and grain yield plant-1 (27.99) (Table 3). Weekly foliar application of GA₃ during the 10–30% panicle heading stage can significantly enhance grain yield in MR219, a widely cultivated Indica rice variety released by the Malaysian Agricultural Research and Development Institute (MARDI).18  This finding underscores the potential of GA₃ application timing in optimizing yield outcomes for hybrid rice production.

 Similarly, in case of δ2p some metrical traits showed the significant higher values viz.  number of grain panicle-1 (85.26), number of fertile grain panicle-1 (4.40) and grain yield plant-1 (28.17). In all the cases the δ2e were found to be very lower values except grain yield plant-1 (0.18). In our study, the phenotypic coefficient of variation (PCV) was consistently higher than the genotypic coefficient of variation (GCV) across all assessed traits. Relevant observations were also reported.19-20 δ2p was higher than that of δ2g in all the characters in our case (Table 3). All the correlation values (Table 4) were found to be positively correlated and significant at 1% which ranges from 0.6108 to 0.9570. A strong positive and significant correlation was observed between the number of fertile grains panicle -1and 1000-grain weight (r = 0.9570). Grain yield plant-1 has been reported to be significantly influenced by the number of grains panicle-1, highlighting the importance of these traits in improving overall yield.21 The above result suggested the possibility of selection of one of the above component character would result in the improvement of other characters.  Correlation values are graphically represented in Figure1.

Various treatments have different role in their mode of action. The rice was most sensitive to sulfonamide. Similar to bacteria, plants possess a folate biosynthetic pathway that plays a crucial role in their growth and development. This pathway is essential for various physiological processes, including DNA synthesis, repair, and methylation.

Sulfonamide antibiotics can interfere with this pathway, thereby affecting plant growth and development by inhibiting folate biosynthesis, a key process essential for plant vitality.22 Gentamycin was utilized as a low-cost alternative to gibberellic acid (GA₃).23 Its potential to replace GA₃ in enhancing plant growth and hybrid seed production makes it a viable option for more cost-effective agricultural practices. Gentamycin, an aminoglycoside antibiotic, exerts its antibacterial effect by inhibiting protein synthesis through binding to the bacterial ribosome. This mechanism disrupts cellular processes essential for bacterial growth and survival. In addition to its medical applications, gentamycin is also used for various agricultural purposes in countries such as Mexico and the USA, demonstrating its versatility in managing plant health and productivity. Plants uptake nitrogen in the form of nitrates and ammonium and molybdenum play an important role in this process and also work as a catalyst in many enzymatic activities.24  In case of nickel compound it is evident that nickel is an essential metal and plays an important role in plant metabolism.  The potential of penicillin and certain heavy metal compounds as promising alternatives to traditional methods for enhancing panicle emergence in CMS (A-line) rice plants which  suggest valuable implications for developing cost-effective agricultural practices and optimizing hybrid rice seed production .25

In this experiment, treatment-2 (Penicillin) outperformed other alternatives to GA3 due to its lower cost, consistently yielding superior results across the cropping season. Penicillin significantly promoted shoot elongation in rice seedlings. Additionally, seedlings treated with penicillin maintained higher levels of nucleic acids and proteins, indicating a positive impact on both growth and metabolic processes.26 Penicillin has been shown to enhance chloroplast pigment formation in intact rice seedlings27, affecting leaf morphology, stem circumference, and internode length.28 A concentration of 400 mg/L is effective in modifying leaf morphology in Glycine max and increasing leaf area in Chrysanthemum. There is a scope of future research to standardize the dose of penicillin for panicle emergence in IR58025A (CMS) line rice for maximum hybrid rice seed production. Penicillin also promotes stem circumference and internode length through cell elongation, likely by increasing levels of gibberellin and cytokinin-like substances, as observed in mungbean seedlings.29 From this result it is evident that the treatment penicillin and GA3 were given more or less similar though penicillin effect was indicated a little bit better than that of GA3 effect.  Penicillin as the most promising treatment, yielding optimal panicle emergence results at a lower cost while significantly enhancing crop output. GA₃ and gentamycin also demonstrated strong efficacy, supporting their role as effective treatments. Micro-histological analysis  of CMS line rice (IR58025A) using SEM provided further insights into treatment impacts on plant structures.30 Notably, penicillin treatment induced a marked loosening of the endothecium layer in the androecium and strengthened the gynoecium, promoting a more fibrous and gametophytic structure. These microstructural modifications indicate an enhanced seed set potential, underscoring penicillin’s viability in improving F1 hybrid rice production.

Conclusion

Among the ten alternative treatments, penicillin emerged as the top performer, offering a compelling combination of high yield and cost-effectiveness. Notably, penicillin’s performance was on par with GA3, with a slight advantage. Potassium dichromate also demonstrated promising results in promoting panicle emergence. Thereafter, remaining eight treatments are performing according to their mode of action. The role of penicillin as a growth regulator presents a promising avenue for agricultural applications, brightening the prospects for its broader use in crop enhancement. The characters showing positive and significant correlation will help to improve yield. In this case further advancement of lines or genotypes can be done based on these character combinations will definitely leads to the development of superior variety with higher yielding ability.

Acknowledgements

The authors express their sincere gratitude to the University of Burdwan and the Rice Research Station, Chinsurah, Hooghly, for their invaluable support and assistance throughout this study.Authors also express deep sense of gratitude to Dr. Jagatpati Tah, for providing valuable guidance as and when needed for this study.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article..

Conflict of Interest

The authors do not have any conflict of interest

Data Availability Statement

The manuscript incorporates all datasets produced or examined throughout this research study.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

his study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials

Author Contributions

Riya Pal : Monitored field trials and execution, Data collection and analysis, Manuscript preparation.

Chand Kumar Santra: Conceptualization, Field layout, Methodology and Correlation  analysis. 

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