Screening Technique Based on Seed and Early Seedling Parameters for Cold Tolerance of Selected F₂-Derived F₃ Rice Genotypes under Controlled Conditions

Abstract

The cold tolerance studies were carried out in a bi-parental F₂ population of a cross between tolerant and susceptible parents (SKUA-529 and HEERA, respectively). The purpose was to screen the individuals of a population for primary cold-tolerance-related attributes. The information generated has a direct application and use in identifying cold tolerance quantitative trait loci (QTLs) and further can be used for genotyping with an appropriate marker system. The screening was carried out on F₂-derived F₃ seeds and F₃ plants for seedling and agronomic traits, respectively. Two tests measuring cold tolerance were conducted. In experiment I, seeds were germinated for 28 days at 13 °C and 7 days at 28 °C, and in experiment II, the seeds were germinated for 72 h at 28 °C, 96 h at 13 °C, and once more for 72 h at 28 °C. Coleoptile length, germination percentage, and radical reduction percentages were all measured in experiment I. The radicle and coleoptile regeneration in experiment II were measured after the cold period. The improvement in cold tolerance was achieved through radicle regrowth, as evidenced by the difference between the second and first measurements. The individual lines from the F_2:3 population that recorded high germination (%) were #21, #13, #14, and #15. The percentage of coleoptile length (PERCOL %) was observed to be between the ranges of 23.33% to 53.00%. The reduction in coleoptile length (REDCOL %) was also obtained, and there was less reduction in #15, #16, and #14 and it had a range between 38.46% and 75%. Radicle regrowth (REDRAG) was high at 13 °C in #7, #11, #30, #35, and #36. Survival of the seedling range was between 33.33% and up to 100%, and the highest survival rate was observed in #16. The main objective of this rotation in temperature was to emulate field conditions where there has been a drop in temperature. The evaluations were done for primary cold stress tolerance traits, and it was found that most of these traits exhibited high variability. The mapping population developed may be utilized to generate a linkage map and locate QTLs for tolerance to cold stress in rice. Further, the identified donors for cold tolerance may be utilized for breeding programs aimed at the transfer of low-temperature stress tolerance into susceptible backgrounds. In general, a genotype with improved seedling germination rates, growth rates, and leaf yellowing scores; high seedling survival; lesser reduction in coleoptile length and in radicle development; and recovery following a cold shock at the seedling stage demonstrated its cold resistance. Genotypes with a low germination percentage, a greater number of days to germination, slow growth rate and higher leaf yellowing score, high reduction in coleoptile and radicle growth, and reduced seedling survival indicated cold susceptibility.

1. Introduction

One-third of the world’s population is fed by rice, making it one of the most significant cereal crops. The estimated increase in the world’s rice consumption is expected to be from 723 million tons in 2015 to 763 million tons in 2020 and 852 million tons in 2035. In India, rice is grown on an area of 43.86 million ha, with productivity of roughly 2390 kg/ha and a production level of 104.80 million tons [1]. It is grown in a variety of climatic and soil environments. Compared to the productivity levels of many other nations, India’s rice production is relatively low. Additionally, marginal, small, and medium farmers own about 90% of the cultivated land, which is another barrier to raising the country’s rice yield. China has the highest productivity (6710 kg/ha), followed by Vietnam (5573 kg/ha), Indonesia (5152 kg/ha), Thailand (4610 kg/ha), and Bangladesh (4375 kg/ha) [2]. The improvement of potential and production yield would be facilitated by the incorporation of biotic and abiotic stress tolerance mechanisms. Wild rice varieties are excellent sources of beneficial genes, not just for yield but also for qualities such as disease resistance and stress tolerance. Hybrid rice cultivation has the potential to boost productivity and the need to develop primitive rice varieties/hybrids ideally suited for cultivation under stress conditions is also a viable option for increasing the overall rice production. About 1 million hectares of hill areas in the hill states of Jammu and Kashmir, Uttaranchal, and the northeastern part of India are used to produce rice under cold stress, which accounts for 2.3 percent of all rice-growing land there [3]. In comparison to the average national output of 9.1 t/h, the average return of this cold-region rice is roughly 1.1 t/h. Low temperature, blast, a dry period, and a relatively short cropping season are some of these places’ main productivity restraints. The cold-temperature stress in such places mostly impacts rice at the early stages and infrequently at the flowering phases as well, leading to sterility and a drastic drop in yield. Low-temperature-induced yield loss is a global issue since rice is a temperature-sensitive field crop [4]. In contrast to other Indian states, Jammu and Kashmir produces only one crop of rice in a season, which is consumed extensively as a staple meal. Even though the majority of the province’s residents depend heavily on rice crops for their livelihood, just 0.27 million hectares of the state’s land is planted with the grain [5]. Rice productivity in the state is high compared to the national average productivity. The total annual rice production in the state is more than 0.59 MT. The land planted with rice is divided into two zones, i.e., Jammu and Kashmir, of which about 40% is in the Jammu division and 60% is in the Kashmir division. Low spring and fall temperatures in the Kashmir valley limit the growing season of the rice crop. Under field conditions, the temperature cannot be readily changed, but seeding time may be changed to match the demands of each physiological stage of the crop growth cycle [6]. The present study was carried out to screen temperate rice of F₂-derived F_2:3 population at the seedling stage for cold tolerance. However, this study will provide correct and precise information regarding cold tolerance before any sort of detailed genotyping is undertaken.

2. Materials and Methods

2.1. Plant Materials and Experimental Conditions

The rice genotypes kept at the Mountain Research Centre for Field Crops (MRCFC) Khudwani, of the Sher-e-Kashmir University of Agriculture Science and Technology of Kashmir (SKUAST-K) provided the seed material. An F₂ mapping population of forty-six individuals resulting from the cross SKAU-529 x Heera was evaluated at the seedling stage, for cold tolerance. Evaluation of individuals in the F₂ and F_2:3 population was performed during the years 2015 and 2016.

The seeds were sanitized using ethanol 70% for 30 s, sodium hypochlorite 1% over 20 min, and followed by distilled-water washing to remove the sticky surfaces. They were put on Petri dishes with two layers of filter paper that had been wetted with distilled water, and later, the wrapped papers were transferred to plastic trays and kept in a growth chamber for germination. Three replications of ten seeds each were used in the experiment, which was run according to a completely randomized design. The radicle lengths of the seeds that germinated at 13 and 17 °C were recorded on a weekly basis for four weeks, and a single reading at 28 °C was collected at the conclusion of the experiment to evaluate with Cruz and Milach’s stress regimes [7].

The seed and seedling quality observations for different component traits of rice genotypes were recorded at different temperatures 28 °C (control) and 13 °C (cold stress).

2.1.1. Phenotyping Screening

According to ISTA regulations [8], the laboratory test for germination was carried out using the between-paper technique. The germination test had three replications with ten seeds in each treatment, which were placed in a seed germinator/growth chamber and kept at constant temperatures of 28 ± 0.5, 13, and 17 °C separately with suitable humidity levels. The earliest and last readings were taken on the fourth and seventh days at 28 °C, as well as at periodic times during cold stress conditions at 13 and 17 °C, respectively. The number of seeds that germinated were counted on the day of the final reading, and the percent of germination and seedling survival were computed as follows.

$$\mathrm{Germination} \mathrm{percent}=\frac{Number of normal seedlings}{Total number of seedlings planted}\times 100$$

(1)

$$\mathrm{Seedling} \mathrm{survival} \mathrm{rate} \%=\frac{Surviving Seedlings}{Sprouting Seedlings}\times 100$$

(2)

2.1.2. Percentage of Seeds with Coleoptile Larger than 5 mm (PERCOL)

The PERCOL was determined using the method provided by [9], taking into account all of the seeds that had germinated 28 days after the start of the cold treatment and checking the proportion of those that had coleoptiles longer than 5 mm.

PERCOL = Number of seeds germinated with coleoptile > 5 mm × 100

(3)

2.1.3. Percentage of Reduction in Coleoptile Length (REDCOL)

The REDCOL was determined by comparing the average coleoptile length obtained 7 days after germination at 28 °C with that obtained 28 days later at 13 and 17 °C (cold treatments) and using the formula provided by [10] to calculate the percentage of coleoptile length reduction caused by germination under cold conditions.

$$\mathrm{REDCOL}=\frac{Coleoptile length under Control-coleoptile length under stress}{Coleoptile length under Control}\times 100$$

(4)

where the average of 10 seeds tested for each replication and genotype determines the coleoptile length.

2.1.4. Radicle Regrowth (RADREG)

Radicle length was measured twice: at the end of the first period of 96 h at 13 °C (LENGTH 1) and at the end of the second period of 72 h at 28 °C (LENGTH 2). According to the equation: RADREG = (LENGTH 2) − (LENGTH 1), where radicle length is the average of the 10 seeds assessed in each replication, two radicle lengths were acquired to allow for radicle regeneration. Radicle regrowth was used in this experiment to evaluate the subjects’ tolerance to cold, as evidenced by the difference between the second and first measurements. The purpose of this temperature rotation was to imitate the temperature variance in field situations.

Radicle regrowth (RADREG) = (LENGTH 2) − (LENGTH 1)

(5)

2.1.5. Statistical Analysis

Tukey’s HSD tests found statistically significant differences between the parents when the percentage of plant survival was examined using an ANOVA in a totally randomized design with a variable number of replications (p = 0.05). For the features of germination percentage at varying temperatures and percentage of reduction in coleoptiles (REDCOL) at 13 and 17 °C, respectively, the analysis of variance indicated very significant mean squares across the genotypes. Each parameter’s analysis was done independently. According to Panse and Sukhatme’s recommendations [11], the analysis of variance was carried out. A chi-square test was run on the data after the F₂ populations were assessed by counting the number of surviving and non-surviving plants in each. Using Pearson’s coefficient correlation, relationships between the measured attributes were found. All analyses were performed in the Statistical Analysis System, SAS Institute, 2000 [12].

3. Results

In the state of Jammu and Kashmir, rice is grown at up to a height of 2200 m above sea level under complete and assured irrigation along the foothills of coniferous forests [7]. The natural streams serve as a source of water for farmers’ fields. Low water and soil temperatures become a major limiting factor in deciding higher yields and early maturity. A repository of temperate rice germplasm is being maintained by SKUAST-Kashmir, which comprises indigenous and exotic collections. The germplasm may be a valuable resource and repository of important alleles for multiple biotic and abiotic stresses. The characterization of such germplasm becomes a priority in order to establish the donors of genotypes that really are cold tolerant so they can be used in breeding initiatives in the future. At the same time, mapping populations serve as a valuable resource for a breeder and a geneticist to locate important genes/QTLs that confer resistance to biotic/abiotic stresses. The phenotyping of such mapping populations needs to be performed with great precision. In earlier research, F₂ or F_2:3 populations have been used successfully to map the genes for various traits [13,14,15].

3.1. Evaluation of Parents for Cold-Tolerance-Related Traits

The germination percentage for SKUA-529 at 28 °C (Control) was 100%, followed by 90.00% (13 °C) and 96.66% (17 °C), which was significantly higher than that for Heera at 28 °C (96.67), followed by 60.00% at 13 °C and 73.33% at 17 °C. The maximum mean difference between control and cold was recorded in HEERA at 13 °C, and less reduction was observed in SKUA-529 at both temperatures. The percent decline in the germination of Heera recorded at 13 °C was 38% followed by 24% at 17 °C, which was significantly higher in comparison to that of SKUA-529. The REDCOL was high in Heera (37.93%) at 13 °C followed by 24.14% at 17 °C, while the values recorded for REDCOL in SKUA-529 under similar conditions were 10.00% and 3.34%, respectively. The high REDCOL indicates the susceptibility of rice seedlings to low temperature at the germination stage. Among the parents, SKUA-529 exhibited high radicle regrowth at 13 and 17 °C. Similarly, the maximum seedling survival rate was observed in SKUA-529 at 15- and 30-day intervals (

Table 1. Evaluation of parents (HEERA and SKUA-529) for cold tolerance.

Characters	Treatment	HEERA	SKUA-529
Germination percent	28 °C	96.67	100.00
	13 °C	60.00	90.00
	17 °C	73.33	96.66
Difference between control and cold germination	13 °C	36.67	10.00
	17 °C	23.34	4.00
Percent decline in germination	13 °C	38	11.11
	17 °C	24	3.34
Reduction in coleoptile (%) (REDCOL)	13 °C	37.93	10.00
	17 °C	24.14	3.34
Radicle regrowth	96 h at 13 °C (L-1)	1.0	1.5
	72 h at 28 °C (L-2)	1.9	2.3
	Regrowth	0.9	1.6
Seedling survival (%)	15 days	80.0	93.33
	30 days	53.3	76.66

GP = germination percent, PERCOL = proportion of seeds with coleoptiles longer than 5 millimeters, REDCOL = decrease in coleoptile length, RADREG = radicle regrowth, and SSP = seedling survival percent.

).

3.2. Evaluation of the F_2:3 Mapping Population for Germination and Seedling Parameters under Controlled Conditions

Seed germination under control and stress conditions was measured with respect to coleoptile size on a weekly basis for 28 days at 13 °C and for 7 days at 28 °C. Only those seeds that had a coleoptile length of more than 5 mm were taken into consideration. In the F_2:3 population, only two temperatures, viz, 28 and 13 °C, were considered for screening of cold tolerance as considerable reduction in germination at 13 °C was observed during the evaluation of rice genotypes as depicted in Figure 1.

3.3. Germination Percentage of the F_2:3 Population

The highest germination percentage of 96.67% was observed in individuals with plant IDs #7, #12, #26, #36, and #41 followed by 93.33% in #4, #11, #19, #20, #28, #30, #37, #39, and #45. About 90% germination was observed in plants #5, #6, #9, #24, #25, #32, #38, and #46. The comparison of germination percentage of the control (28 °C) with that of the F_2:3 population under cold temperature (13 and 17 °C) showed a reduction of more than 50% in genotypes #26, #30, and #36 (66.66%), which indicated their cold susceptibility at the germination stage.

The genotypes that showed less difference in germination under cold were #21 (26.67%) followed by #13, #14, and #15 (33.34%), which indicated better tolerance toward cold as depicted in Figure 1.

3.4. Percent Decline at Two Different Temperatures

The decline in germination percentage among the individuals of the population showed a range of 26.67 % to 66.67 % when comparing cold-stressed plants against control. The germination percentage recorded at 13 °C was the highest in #15 followed by #23 and #16, and the lowest was achieved in #10 followed by #33, #5, and #9. The high germination percentage under cold treatment predicts tolerance to cold. The minimum germination percentage at 13 °C was 23.33% and the maximum was 50.00%, showing tolerance and susceptibility against cold.

3.5. Percentage of Seed with Coleoptile Length > 5 mm (PERCOL)

A wide difference was observed among the individuals of the population for PERCOL. Only those seeds were considered whose germinated seeds had a coleoptile length >5 mm. The PERCOL was highest in #15 (53.33%) followed by #16 and #40 (46.67%) and #18, #34, and #39 (43.33%).

3.6. Reduction in Coleoptile Length (REDCOL)

There was a significant difference among the individuals of the F₂ population for REDCOL. Coleoptile length decreased by a small amount in five individuals, viz., #15 (38.46%) followed by #16 (41.67); #14, #23, and #39 (50.00%); and #42 (52.39). The increase in REDCOL depicts susceptibility to cold and a lower REDCOL reveals resistance against cold.

3.7. Radicle Regrowth of the F₂ Population

In field circumstances, there is typically a chilly interval between hot intervals, which causes cold-stress damage. Experiment II was conducted to simulate field circumstances, and a significant difference was observed in the rice genotypes with respect to radicle regrowth rate. Radicle length was measured twice: once at the conclusion of 96 h at 13 °C (Length 1) and again at the end of the second period of 72 h at 28 °C (Figure 2). Radicle regrowth for all genotypes ranged from 0.2 to 2.0 cm. As shown in

Table 2. Effect of cold stress on seedling parameters of rice genotypes.

Genotypes	Germination % under Control	PERCOL	REDCOL
MEAN	86.59	34.49	61.87
SD	8.39	6.67	8.26
CV (%)	9.69	19.35	13.36

Based on the ANOVA and Tukey’s HSD tests, the findings were statistically significant (p = 0.05). Separate analyses were run for each parameter. PERCOL stands for the percentage of seeds having coleoptiles larger than 5 mm. REDCOL: coleoptiles’ length reduction percentage.

, several genotypes had exceptional radicle renewal abilities under cold stress, and the individuals #22, #32, and #34 of the F₂ population had the highest radicle regrowth (more than 2.0 cm) while the lowest (less than 5 mm) was observed in #26 and #37.

3.8. Percentage of Survival of Seedlings after 15- and 30-Day Intervals

To screen out the survival of seedlings, the F_2:3 population was subjected to a low temperature at the seedling stage. At 15 days after sowing (DAS), under stress conditions, the highest seedling survival percent was recorded in the lines #16, #19, #21, #22, and #31 (100%), but the survival rate decreased, and mortality increased with the increase in time duration. Lowest values were observed in plant IDs #34 (33.3%), #44 (40%), and #32 (40%). The number of seedlings in the growth chamber decreased under controlled settings during the seedling stage due to low temperatures. At 15 DAS, at 13 °C, low-temperature seedling mortality was seen in the F₂ population. At 30 DAS, most of the plants showed a reduction in seedling survival, which was below 50%. The highest reduction was in #12 (20%) followed by #30 (20%) and #34 (23.3%) (

Table 3. Seedling survival (%) of F_2:3 populations after 15- and 30-day intervals.

F2:3 Population	Seedling Survival (%) 15 Days	Seedling Survival (%) 30 Days	F2:3 Population	Seedling Survival (%) 15 Days	Seedling Survival (%) 30 Days
1	66.7	40.0	24	80.0	73.3
2	80.0	66.7	25	83.3	80.0
3	83.3	80.0	26	83.3	70.0
4	86.7	60.0	27	66.7	50.0
5	80.0	40.0	28	80.0	40.0
6	46.7	43.3	29	100.0	93.3
7	83.3	83.3	30	43.3	20.0
8	40.0	26.7	31	100.0	83.3
9	46.7	40.0	32	40.0	23.3
10	63.3	46.7	33	83.3	46.7
11	83.3	80.0	34	33.3	23.3
12	40.0	20.0	35	66.7	60.0
13	66.7	43.3	36	83.3	63.3
14	60.0	53.4	37	66.7	40.0
15	66.6	33.3	38	86.7	60.0
16	100.0	90.0	39	63.3	43.3
17	86.6	80.0	40	60.0	50.0
18	80.0	60.0	41	66.7	43.3
19	100.0	86.6	42	40.0	40.0
20	60.0	53.4	43	50.0	40.0
21	100.0	93.4	44	40.0	26.6
22	100.0	96.6	45	60.0	50.0
23	66.6.0	40.0	46	86.7	66.7

Screen out F_2:3 population for testing its survival under low temperature at the seedling stage after 15 and 30 DAS.

).

4. Discussion

The cold-tolerance studies were carried out in a bi-parental F₂ population of a cross between tolerant parent SKUA-529 and susceptible parent Heera. The purpose here was to screen the individuals of a population for their primary cold-tolerance-related attributes. The information generated has direct applicability and use in the mapping of QTLs for cold tolerance and genotyping using an appropriate marker system was carried out. For our purpose, the screening was carried out on F₂-derived F₃ seeds and F₃ plants for seedling and agronomic traits, respectively. The conclusions drawn in this way qualify a progenitor F₂ plant for the respective traits. Amongst 46 F_2:3 plants, plants #21, #13, #14, and #15 were the best for rapid germination and high germination percent. Yoshida [16] reported the greatest influence of temperature on germination. Cruz and Milach [9] found that low-temperature stress may impact rice seed germination, preventing growth at the seedling stage. Early seedling germination and quick development take place in direct seedling culture at low temperatures. The authors of [17,18] tested 68 rice germplasms for cold tolerance during the germination stage and found that the rate of germination, the length of the radicle, and the coleoptile all decreased in the cold, which may be due to decreased metabolic activity and inactivation of enzymes that are essential for germination. Under low temperatures, plants #15 and #16 were superior with respect to coleoptile growth, while plant numbers #7, #11, #30, #35, and #36 performed well for radicle regrowth. However, better seedling survival under suboptimal growth conditions was recorded in plants #16, #19, and #21. The radicle growth values varied from 0.2 to 2.0 cm, demonstrating that various genotypes had very distinct germination processes following the cold period. The individuals #22, #32, and #34 of the F_2:3 population had the highest (more than 2.0 cm) and #26 and #37 had the lowest level of radicle regrowth (less than 5 mm), respectively. This was in conformity with the results of Farzin [19], who identified that the genotypes with the highest radicle regrowth of more than 18 mm were PR27137-CR153, Khazar, Hasani, and Gil2. Coleoptile regrowth of more than 18.5 mm was observed for PR27137-CR153, Khazar, and Hasani, which was the highest. Yoshida [20] showed that the coleoptile and radicle activation and growth stages are affected when temperature has an impact on the germination stage. It is possible that the low temperature’s direct impact on cellular elongation and division during these phases is what is causing the reduction in coleoptile development, or that it is also having an indirect impact that is causing a metabolic imbalance [21]. An important predictor of a crop’s tolerance to freezing stress is the measurement of radicle regrowth [22,23]. Low temperature leads to the rapid escape of solutes such as amino acids and carbohydrates from the seeds due to the dry seed’s incomplete plasma membrane and the disruptions brought on by its regeneration during the imbibition phase at low temperatures. The cellular membrane is considered to be a significant site of damage from cold and the biggest factor behind various metabolic issues seen in cells [19]. Differentiations in the lipid layer’s composition were thought to be the source of the higher tolerance to the phase transition at low temperatures, with the theory being that the higher the lipid layer’s degree of saturation, the lower the temperature at which phase transition could occur [24]. Low temperatures during the seedling stage resulted in a decrease in the number of seedlings in the growth chamber under controlled circumstances. At 15 DAS, at 13 °C, low-temperature seedling mortality was observed in the F₂ population. At 30 DAS, most of the plants showed a reduction in seedling survival, which was below 50%. The highest reduction was in #12 (20%) followed by #30 (20%) and #34 (23.3%). Similar findings were found, and it was hypothesized that genotypes with strong germination and seedling vigor under low-stress circumstances are also probably more resistant to low-temperature exposure during the booting and blooming stages. Cold-stress damage in rice can occur at all growth stages. The content of damage depends on stress, cold duration, rice ecotype, and variety. Bertin [25] reported a positive correlation between early seedling cold tolerance with flowering stage cold tolerance. According to Akhtamov’s findings [26], the insertion of advantageous O. rufipogon alleles might speed up the development of rice cultivars with high levels of coleoptile elongation and low-temperature germination (LTG) in japonica cultivars. The outcomes demonstrated that the effects of low temperature differed across genotypes investigated in both studies. In SKAU-402, followed by SI-6, K-332, SI-5, and SI-3, the highest germination was attained with the least percent reduction in germination, i.e., 0.13, 0.17, 0.24, and 0.32 at 13 °C [27]. Low temperatures damage rice plants during their germination, vegetative development, and reproductive phases. To determine the degree of cold resistance under artificially produced low temperatures at the seedling stage, 39 rice genotypes, including 36 near-isogenic lines (NILs) of BRRI dhan29, were assessed [28].

5. Conclusions

The Cruz and Milach’s guidelines for seed and seedling evaluation were used to assess the genotypes of rice for their ability to withstand cold (2004). In a germinator in the lab, seeds were germinated under two conditions: 28 °C for 7 days (control) and 13 °C, 17 °C for 28 days (cold). In general, a genotype’s tolerance to cold was indicated by higher germination rates, faster growth rates, lower leaf yellowing scores, higher seedling survival rates, lessening reductions in coleoptiles length, lessening reductions in radicle growth, and regrowth following a cold shock at the seedling stage. Low germination rates, long germination times, sluggish growth rates, high levels of leaf yellowing, low levels of coleoptile and radicle development, and low seedling survival indicated susceptibility to cold stress. Overall, in this study the genotypes possessing cold tolerance were identified; these genotypes can be used as parental lines for generating germplasm possessing tolerance to cold-temperature stress. The phenotypic evaluation of an F_2:3 population has helped to generate basic data for its further use in studies aiming at the identification of QTLs/genes governing cold tolerance. In a breeding effort to create future cultivars of cold-tolerant plants, the genotypes discovered in this study can be employed as parents.