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Utilization of anther culture technique for rice improvement in the Philippines

Victoria C. Lapitan1, Ernesto B. Cayaban2, Leslie T. Roferos1, Genaro O. San Valentin3 and Leocadio S. Sebastian4

1PhilRice-Los Baos, Pili Drive, College, Laguna 4031, Philippines, Email vicky@laguna.net
2
Department of Agronomy, College of Agriculture, UP Los Baos, College, Laguna 4031, Philippines
3
Rhodas Subdivision, Los Baos, Laguna 4030, Philippines, Email gsanvale@hotmail.com
4
Philrice-CES, Maligaya, Science City of Muoz, Nueva Ecija 3119, Philippines Email lsebastian@philrice.gov.ph

Abstract

Biotechnology can certainly provide solutions to most problems associated with breeding for rice improvement. Anther culture technique offers great opportunities for accelerating breeding progress and generating as well as directing variation to increase genetic diversity useful for rice improvement.

Through anther culture, we have generated 10 doubled haploid lines (DHLs) from F1-506, a cross between C4044-2B-2-2 and IR13540-56-3-2-1. Further evaluations of these lines both under screenhouse and field conditions resulted to selection of three (3) outstanding DHLs. Aside from giving stable performance, these lines were selected for their high phenotypic acceptability, high yield, and uniformity. Likewise, results of grain quality evaluation revealed that the selected DHLs were better than the parents. On the succeeding replicated yield trials (RYT), from R4 to R6 generations, these elite DHLs consistently outyielded the check varieties, IR72 and PSBRc 18. The results connote that anther culture technique shortened the breeding cycle as we have generated and developed elite breeding lines within 3 years while conventionally, it takes 6 to 7 years for selected inbreds to reach RYT.

From the other set of experiment, variants were generated from the popular IR64 variety utilizing the technique. Three (3) outstanding DHLs were produced with resistance to different soil-related stresses such as P, K, Zn, S, and Cu deficiencies. Evaluation of these variants showed improvements on their root system in terms of root length and weight as compared to the seed-derived IR64 variety.

Media summary

Elite breeding lines were developed at PhilRice through anther culture within three years which shortened the breeding cycle by 3 years. It broadened the genetic diversity by generating variants useful for rice improvement.

Key Words

Gametoclonal variation, somaclonal variation, regenerants, Minus One Element Technique (MOET)

Introduction

Rice production in the Philippines grew from 4M tons in 1965 to 9.1 M tons in 1985, however, from thereon, the growth declines to 1.1% per year. By the year 2010, the country’s population is expected to grow to 87 M from 61 M in 1990. Yields of rice have to be doubled in the next 20-30 years for the country to adequately feed its population. As the rice productivity continues to decline due to reduced hectarage planted to rice, urbanization, post-harvest loses, and continued emergence of new pests and diseases, the only remaining source of output growth is yield improvement. Therefore, for further increases in rice production, research efforts on rice varietal improvement should be strengthened and aimed at continuously developing different cultivars that will help sustain most of the requirements. Although plant breeding methods have considerably contributed to increasing the productivity of modern rices, advanced technologies to complement conventional breeding procedures must be employed. Tissue culture techniques such as anther culture, gametoclonal and somaclonal variation methods offer great opportunities for accelerating breeding progress, and generating as well as directing variation to increase genetic diversity in rice.

Fixation of new characters in a selected line usually takes several generations. However, anther culture technique was reported to shorten the conventional cycle by 3 to 4 generations. The use of the technique is practical for varietal improvement in rice. It has generated a wide range of genetic variability which resulted in the release of several varieties of rice. A large number of rice varieties with outstanding characters have been developed and which are becoming popular in production (Zhang 1982, Swaminathan 1986, Zhu and Liu 1986, Zapata et al. 1982, Hu 1985)

This project aims to utilize the anther culture technique to complement the conventional breeding methods being done at the Philippine Rice Research Institute (PhilRice) and the University of the Philippines Los Baos (UPLB) to improve yield, grain qualities, resistance to pests and to generate novel variants which will be useful for rice improvement.

Methods

Two separate experiments were conducted in this study. In the first experiment, the F1s generated from the cross between C4044-2B-2-2 (P1) and IR13540-56-3-2-1 (P2), both with exceptional attributes like high yield, tolerance to pests and diseases, and with good grain qualities were used as anther sources. The materials were utilized at the breeding program of PhilRice and UPLB in irrigated lowland condition. The emphasis was to generate elite breeding lines or varieties that will adopt to this ecosystem. In the other experiment, anthers were collected from the popular IR64 variety and were subjected to anther culture.

Anthers were plated into the callus induction medium consisted of N6 + 2.0 mg/L 2,4-D + 1.0 mg/L NAA + 0.5 mg/L Ki + 50 g/L maltose. Induced calli were subcultured into the plant regeneration medium (MS + 2.0 mg/L Ki + 0.5 mg/L NAA + 30 g/L sucrose) and incubated under lighted condition until roots and shoots formed. Regenerated plants were potted out in the greenhouse.

At maturity, seeds (R1 generation) from individual regenerants (R0 generation) were harvested by panicle. Single panicle from regenerants originating from one anther constituted one recombinant inbred line or doubled haploid line (DHL). Evaluations were conducted in both screenhouse and field conditions. All lines were evaluated based on phenotypic acceptability, yield, reaction to pests and diseases and some soil-related stresses. DHLs were also evaluated for grain quality attributes i.e. milling and physical qualities, and their physicochemical characteristics. To determine however, the performance of DHLs under nutrient deficient conditions, plants were evaluated using the minus one element technique (MOET), a kit developed by PhilRice that determines the kind of nutrients limiting to the soil. .

Results

A total of 1,229 anthers from F1-506 were subjected to anther culture. Out of these, 11 plants (R0) were generated and potted-out in the screenhouse. At maturity, only 10 panicles (R1 generation) which were labeled doubled haploid lines (DHLs) were harvested from these plants and then evaluated for their performance both under screenhouse and field conditions.

Table 1 shows the performance of these DHLs at R3 generation. All DHLs surpassed the yield of P1 but not of P2 except for VAC 550 which had the highest yield of 6.32 tons/ha. The yield advantage could be attributed to the highest number of panicle per square meter that this line obtained.

Grain quality evaluation was also performed among the DHLs (Table 2). In terms of milling quality, VAC 550 had the highest head rice yield of 42.4% which was comparable to P2 (45.0%) but better than P1 (25.8%). On the other hand, an improvement in terms of physical quality was observed among the DHLs. Consistent with its performance, VAC 550 produced the longest grains (7.20 mm). No variation was observed in terms of shape while significant variation in terms of translucency for milled rice was observed, with VAC552 having the highest value. All DHLs had intermediate amylose content which ranged from 22.41-22.82 and were comparable to both parents. An improvement in terms of pasting characteristics was also observed. All DHLs were softer in texture as compared to their parents. The results on grain quality analysis connote that the generated DHLs were better than the parents.

From the 10 DHLs, 3 outstanding lines were selected, VAC, 550, VAC 553 and VAC 547, for their phenotypic acceptability, high yield, uniformity, and better grain qualities. The lines were further advanced to replicated yield trial (RYT) for 3 seasons from R4 to R6 generations. These lines showed stable performance and consistently outyielded the check varieties, IR72 and PSBRc 18 during such trials. This work proved that anther culture enhanced the generation and development of elite breeding lines. It took us only 6 generations or 3 years to generate elite DHLs, starting from culturing of anthers to RYT 3. With the technique, the cycle was shortened by 3 years because conventionally, it takes 6 to 7 years for selected inbreds to reach RYT 3. To date, the selected outstanding DHLs are on the pipeline for the National Cooperative Test (NCT), the ultimate test to varietal approval.

In the other experiment, anther culture technique likewise proved to generate new genetic traits for adverse conditions. From 34,240 anthers of IR64 subjected to anther culture, 3 outstanding DHLs (VAC 5, 6, and 7) were generated and developed with tolerance to P, K, Zn, S, and Cu deficiencies. As shown in Table 3, the 3 DHLs were better than the seed-derived IR64 in terms of their agro-morphological attributes. The root system of the selected lines was also examined. Evaluation of these materials revealed that in terms of the total root length, weight, and pattern of distribution, DHLs were improved. Compared with the seed-derived IR64, DHLs had longer, heavier, and robust roots. Likewise, VAC 8 was also found to be resistant to saline condition. The results demonstrated that anther culture could generate variants that will be useful in rice improvements. Several unique variants are produced by somaclonal and gametoclonal variations [DH somaclones] and the methods proved to be much shorter than the conventional one. To date, these 3 DHLs are included to the pool of germplasm being utilized by the plant breeders at PhilRice and UPLB for improvement of varieties in adverse conditions.

Table 1. Field performance of the generated DHLs at R3 generation

DH lines

Yield
(ton/ha)

Plant height
(cm)

Productive tillers
(%)

No. of panicle per sq.m

Panicle length
(cm)

Filled grains
(%)

VAC 547
VAC 548
VAC 549
VAC 550
VAC 551
VAC 552
VAC 553
VAC 554
VAC 555
VAC 556
Parent 1
Parent 2

5.63
5.16
5.07
6.32
4.82
4.96
5.17
5.33
5.06
4.59
3.64
5.82

131.1
125.0
126.2
127.4
129.3
130.6
127.6
128.8
124.3
130.1
99.5
94.2

86.8
80.8
83.6
80.8
87.0
84.
86.5
83.9
86.6
81.8
79.4
85.5

294.4
296.0
294.4
340.8
299.2
304.0
316.8
305.6
289.6
308.8
283.2
310.4

23.5
24.9
23.8
24.1
23.6
23.7
23.9
23.7
24.3
22.8
23.3
24.7

83.5
65.0
82.7
81.7
86.4
75.2
80.4
68.5
72.1
78.8
86.8
70.8

Table 2. Grain quality analysis of the generated DHLs at R3 generation

DH lines

Milling quality

Physical quality

Physicochemical characteristics

Milled rice (%)

Head rice (%)

Translucency

Grain size (mm)

Amylose content (%)

Alkali spreading

VAC 547
VAC 548
VAC 549
VAC 550
VAC 551
VAC 552
VAC 553
VAC 554
VAC 555
VAC 556
Parent 1
Parent 2

64.9
64.3
66.3
65.9
68.4
67.7
63.7
65.5
65.1
65.5
69.0
66.0

34.8
37.6
31.7
42.4
37.3
30.1
35.4
34.6
34.2
37.6
25.8
45.0

30
66
54
57
64
84
72
76
69
64
92
79

6.9
7.0
6.9
7.2
6.9
7.0
6.9
7.0
7.0
7.0
5.9
6.8

23.6
23.8
24.0
22.9
23.4
23.1
23.3
23.5
22.7
22.4
22.2
23.3

6.9
7.0
7.0
7.0
7.0
7.0
6.8
7.0
7.0
7.0
7.0
6.9

Table 3. Agro-morphological and root characteristics of the generated variants and the seed-derived IR64

Traits

VAC 5

VAC 7

VAC 8

SD-IR64

Plant height (cm)
No. of panicle
No. of tiller
Panicle length (cm)
No. of filled grains
No. of unfilled grains
Root length (cm)
Root weight (g)

125.30
36.33
43.67
25.7
107.63
38.83
92.10
21.40

137.30
22.33
31.0
26.36
78.27
77.63
84.70
15.30

132.00
21.67
33.00
27.98
110.93
43.80
90.00
22.80

119.70
29.00
29.33
24.70
89.47
28.40
71.60
13.40

Conclusion

Anther culture technique enhanced the generation and development of new elite breeding materials useful in rice improvement. In the first experiment, three elite DHLs were generated within 3 years while conventionally, it takes 6 to 7 years to produce purelines through continuous selfing or backcrossing. These lines were developed with stable performance, high yield, and better grain qualities. Likewise, new variants were generated for adverse conditions. Three DHLs were produced with tolerance to P, K, Zn, S, and Cu deficiencies. The technique improved the popular IR64 variety in terms of tolerance to some nutrient deficiencies while retaining all favorable qualities. These new variants are useful sources for genetic variation that could be of value to plant breeders.

References

Hu H. (1985). Use of haploids in crop improvement. In Biotechnology in International Agricultural Research. Proc. Inter-Seminar IARC’s and Biotech. Int.l Rice Research Institute, College, Laguna, Philippines. pp. 763-772.

Swaminathan M.S. (1986). Plant research and world agriculture. Plant Molecular Biology Reporter 4(1):1-17.

Zapata F.J., L. Torrizo, R.D. Romero, and M.S. Alejar (1982). Androgenesis in Oryza sativa. In: Plant Tissue Culture, A. Fujiwara (ed.). Maruzen, Tokyo, pp. 531-532.

Zhang Z.H. (1982). Application of anther culture techniques to rice breeding. In: Proc. on Rice Tissue Culture Planning Conference. IRRI, Los Baos, pp. 55-61.

Zhu D., X. Pan and W. Liu (1986). Developing high quality rice via anther culture of hybrid rice. In: Abstract of the VI International Congress of Plant Tissue and Cell Culture. Minnesota, USA, p. 413.

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