Regeneration of rice double haploids using a onestep culture procedure

J. Plant Physiol.

Vol. 141. pp. 610-614 (1993)

Regeneration of Rice Double Haploids Using a one Step Culture Procedure M.

A.

MARASS!,

O. A. Bovo, G. L.

LAVIA,

and L. A.

MROGINSKI

Facultad de Ciencias Agrarias (UNNE)-mONE (Instituto de Botanica del Nordeste), C.C.209, 3400-Corrientes, Argentina Received January 30, 1992 . Accepted October 25, 1992

Summary

Double haploid plants of rice (Oryza sativa L.) were regenerated using two protocols. In protocol 1, anthers from the variety Colonia Masdas 5 MAG were cultured on basal N6 and B5 medium. Both media were supplemented with 5 % sucrose and different combinations and concentrations of NAA, 2,4-D, KIN and BAP. All media tested induced calli after 15 d in culture, which appeared only from pollen grains. The best response was obtained with N6 + 2.0mg·L-l NAA + O.5mg·L-l KIN. Two types of callus arose from the explants: embryogenic and non-embryogenic. The first type was firm, white, and had a nodular surface. Scutella with coleoptile-like leaf structures at different stages of development were observed. After 30 d in culture, calli were transferred to the light. Within 3 d, shoots turned green from more than 85 % of the explants that had produced white shoots. These shoots continued growing and produced a good root system. In protocol 2, pieces of callus, obtained in the same medium used in protocoIl, were subcultured on basal medium MS (3 % sucrose) with different concentrations and combinations of NAA, BAP, and KIN. Maximum shoot regeneration was achieved with MS + 0.1 mg' L -1 NAA + 1 mg' L -1 BAP, but root development was poor. Shoots without roots were then transferred to basal medium without hormones plus 8 % sucrose; roots then formed and tillering occurred within 15 d. Protocol 1 was successful with seven additional rice varieties. Plants from protocol 1 and 2 were successfully established in soil.

Key words: Oryza sativa, anther culture, haploids. Abbreviations: NAA = naphthalenacetic acid; 2,4-D = 2,4-dichlorophenoxyacetic acid; KIN = 6-furfurylaminopurine; BAP = 6-benzylaminopurine.

Introduction

In recent years anther culture techniques have been developed to produce doubled haploid plants, and new varieties have been released. In rice, progress has been achieved by altering growth conditions of donor plants, providing physical or chemical treatments to the anthers, and by developing new culture media (reviews by Chen, 1986; Yamada and Lob, 1984). The protocol most frequently used to obtain haploids from anther culture of rice involves two steps: callus induction in the dark and plant regeneration where calli were transferred to a second medium and incubated in the light. © 1993 by Gustav Fischer Verlag, Stuttgart

This paper reports the production of doubled haploid plants of rice using two protocols: i) callus induction in the dark followed by plant regeneration, without subculture, in the light; and ii) callus induction in the dark followed by plant regeneration on another medium in light.

Materials and Methods Plant materials

Eight rice cultivars (Oryza sativa L.) were used, five from Argentina: Colonia Masdas 5 MAG., Chajari, Guayquirar6 P.A., L.P.

Regeneration of Rice Double Haploids

611

Table 1: Effect of protocol 1 culture medium on callus induction and plant regeneration of rice anthers, variety Colonia Masdas 5 MAG. Basal

Growth Regulators (mg · L - I)

Anthers

Callus transferred to light

% callus

Green shoots

White shoots %

Medium

NAA

2,4-D

KIN

BAP

cultured

Embryogenic

Non embryogenic

%

N6

2 0.2 0

0 0 2

0.5 2 0

0 0 0

376 537 349

20.5 0 0

0 8.7 17.0

86 0 0

14 0 0

B5

0.5

0

0.5

472

0

12.0

0

0

PROTOCOL

LIGHT

DARK Anther Cui lure .nd

Plant

C.llua Induotlon

RetlMf.llon

--..

111

1

Anther Cultur. ,nd C.llu. Induollon

2

....

C.llue Subculture on

l.4edlum

W Plant

Rlgen.ratlon

1Mf-r.1IJ ... 1iJ RftG~r.tlon

Fig. 1: Protocols employed in the regeneration of rice plants from anther culture.

Itape P.A. and Yerua P.A.; one from India: Basmati-370; one from U.S.A.: Lemmont; and one from Italy: Sequial. Detailed results are presented for Colonia Masdas 5 MAG only. Experiments were conducted during the summers of 1988 and 1989. Sheaves containing panicles were wrapped in plastic bags and pretreated at 8 °C for 8 d. They were then surface sterilized in ethanol 70% (v/v) for 3min and then 20% commercial bleach (1.6% sodium hipochlorite, final concentration) for 20 min. The panicles were then rinsed 3 x with sterile distilled water. Callus induction

The developmental stage of microspores was determined by squashing anthers and staining with acetic carmine. Anthers containing uninucleate microspores were found in the middle portion of the panicles and were cultured on callus induction medium that was composed of the mineral salts of either Chu et al. (1975) (N6) or Gamborg et al. (1968) (B5) medium. Both media were supplemented with 5 % sucrose and different combinations and concentrations of NAA, 2,4-D, KIN, and BAP (Table 1). The pH of the medium was adjusted to 5.8 with KOH and HCI and solidified with 0.8 % agar. Glass flasks (40 mL capacity) containing 10 mL of culture medium each were autoclaved at 120°C for 20 min. Approximately 700 anthers per medium were cultured and each medium was repeated at least three times for one year. Plant regeneration

Calli with an approximate diameter of 0.5 cm had formed by 35 d in culture. Plant regeneration occurred using two protocols (Fig. 1). In protocol 1, calli were transferred at 35 d from dark to light (l4-h photoperiod, 19W· m- 2 provided by cool white fluorescent lamps, 27 ± 1°C). In protocol 2, calli were subcultured at 35 d to fresh me-

dium composed of the mineral salts, vitamins, and sucrose of Murashige and Skoog (1962) (MS) supplemented with different combinations and concentrations of NAA, BAP, and KIN. These calli were incubated as in protocol 1. Green plantlets with roots were transferred to soil and grew in a greenhouse to maturity.

Chromosome counting

Chromosome counts were made before transferring the plants to soil. Root tips were isolated at midday and fixed in 20 mM 8-oxiquinolein for 3 h and subsequently stained using the Feulgen technique.

Results and Discussion

Callus induction All media tested induced calli, which began to appear from the cultured anthers 14 d after culture. Callus formation from tissues other than the pollen grain was never observed (Fig. 2 a), which is in agreement with others (Chang et aI., 1978; Yamada and Loh, 1984; Chen, 1986; Nakano and Maeda, 1989). The best response from the four callus induction media tested occurred on N 6 medium supplemented with 2mg·L- I NAA and O.5mg·L -I KIN (Table 1). This medium gave the highest percentage of anthers with callus. Two types of callus tissue arose from explants cultured on this medium. The first type was compact, somewhat uniform in structure, yellow and not embryogenic. The second type occurred after 2 to 3 weeks of culture. It was firm, nodular, white, and embryogenic (Fig. 2 b). Scutella of somatic embryos with coleoptile-like leaf structures at different stages of development were observed on this medium but not on the other three media evaluated. Our embryogenic and non-embryogenic calli from anther cultures of rice are similar in structure and color to those obtained from other graminaceous species (Vasil and Vasil, 1981; Vasil, 1982; Bovo and Mroginski, 1989). In the dark our somatic embryos grew and assumed different developmental stages (Fig. 2 c). Many of these somatic embryos developed into green plants without having to change media. The high NAA level in this medium in combination with KIN promoted the proliferation of embryogenic callus. In contrast, 2,4-D alone or in combination with NAA, KIN, or BAP did not promote embryogenic callus. The improvement of embryogenic callus by cytokinins has been reported in other graminaceous species as Paspalum (Bovo et aI., 1985; Bovo and Mroginski, 1986).

612

M. A. MARAss!, O. A. Bovo, G. L.

LAVIA,

and L. A. MROGINSK!

Fig. 2 a: Callus formation from rice anthers after 30 d in culture. Fig. 2 b: Embryogenic callus obtained from anther culture on N6 medium plus NAA (2 mg· L-I) and KIN (0.5 mg· L -1) after SO d in the dark. Fig. 2 c: Coleoptile leaf like structure obtained from embryogenic callus incubated in the dark after 60 d of culture. Fig. 2 d: Green rice shoots obtained from coleoptile-like structures after 3 d in the light.

Plant regeneration Protocol 1 After 30 d, calli produced on the four induction media were transferred to light. Callus originated on N6 plus 2 mg· L -I NAA and 0.5 mg · L-I KIN contained white shoots. Upon transfer to the light, 85 % of these shoots turned green within 3 d (Table 1, Fig. 2 d). Green and white shoots occasionally occurred on the same callus (approximately 13 %). The green shoots produced roots (Fig. 3 a). The frequency of chlorophyll deficient plants was about 14% (Table 1). Calli produced on the other media did not form shoots when placed in the light and eventually died. Chu et al. (1976) found that basal medium devoid of hormones produced plants from pollen grains of rice through somatic embryogenesis. However, the inclusion of growth regulators in our study was necessary to obtain a high percentage response.

Protocol 2 Thirty-day-old calli subcultured to MS medium supplemented with different combinations and concentrations of NAA, BAP, and KIN also produced shoots (Table 2). The

best response 50 % of calli producing green shoots, was achieved with MS + 0.1 mg· L -1 NAA and 1 mg· L -I BAP. This result is higher than previously reported (Genovesi and Magill, 1979; Guiderdoni et aI., 1986; Cho and Zapata, 1988). The presence of chlorophyll-deficient plants also had been low (on average, 8.5 %). The combination of NAA and BAP that we used for shoot regeneration had been previously used by Bovo et al. (1985) and Bovo and Mroginski (1986) in Paspalum tissue culture. The other growth regulator combinations tried by us were less effective. Callus obtained from N6 medium plus 2 mg · L-I NAA and 0.5 mg· L -I KIN and subcultured to MS medium plus 0.1 mg· L -I NAA and 1 mg· L -I BAP gave the best response compared with the other callus induction media. This was probably due to the presence of white shoots on the callus prior to subculture. Rooting was poor when protocol 2 was used. To promote rooting, shoots were subcultured on basal medium MS with 8 % sucrose and no hormones. Many roots formed at the base of the shoots, and abundant tillering was observed after 15 d. This result is in agreement with Maretzki and Hiraki (1980) in sugarcane, and Bovo and Mroginski (1986, 1989) in Paspalum, where higher levels of sucrose promoted rooting. Anthers from seven additional rice varieties were cultured on N6 basal medium supplemented with 2mg·L-I NAA

Regeneration of Rice Double Haploids

613

Fig. 3 a: Root formation from green shoots after 20 d in the light and 80 d in anther culture. Fig. 3 b: Heading of anther-culture-derived plants of rice grown in the greenhouse for 140d. Table 2: Interaction between callus induction and shoot regeneration medium on anther-derived callus of rice, var. Colonia Masdas 5 MAG. after 35 days in culture. Shoot Regeneration medium**

Callus induction medium

1 BAP*

1 BAP" + 0.1 NAA* Basal Medium

No of

Growth regulators NAA

N6

2 0.2 0

B5

0.5

2,4-D 0 0 2

KIN

BAP

cultures

Callus with shoots %

Cultures

Callus with shoots %

1 KIN* + 1 NAN'

Cultures

Callus with shoots %

Green

White

No

Green

White

No

Green

White

39 15 30

26.7 0 3.6

23.2 0 26.4

35 15 35

17

0 0

25

0

0

20

0.5 2 0

0 0 0

36 20 35

50 5 14.2

9.9 5 12.3

0

0 .5

25

0

0

o o o

0

o

* mg ' L- 1; ,'* Basal medium Murashige and Skoog (1962) (MS). and 0.5 mg . L - 1 KIN (Table 3). The results obtained were similar to those presented for Colonia Masdas 5 MAG. From 30 to 70 % of the anthers from the indica rice varieties Basmati-370 and Lemmont produced green plants, which is an improvement over the result of Raina et al. (1987) with Basmati-370. Genotype is an important factor for anther culture success in rice (Abe and Futsuhara, 1986; Miah et al., 1985; Mikami and Kinoshita, 1988). Many japonica cultivars show a high capacity for callus production and plant regeneration when exposed to low concentrations of growth regulators. However, indica varieties have displayed a low capacity for both responses, even with high hormone concentrations. This variation may by endogenous factors (Sundaru et al., 1983). Our anther culture results were satisfactory when protocol 1 was used with eight rice varieties in use in Argentine today. The use of protocol 1 for anther culture on F1, F2 and F3 hybrid progenies has given excellent results (data not shown). Chromosome counts from root tips of plants regenerated (N6 plus 2 mg' L - 1 NAA and 0.5 mg' L-1 KIN) with or without subculture to a second medium indicated that 25 % were diploid, which is in agreement with the results of Guha-Mukherjee (1973), but not with Geno-

Table 3: Callus induction and shoot regeneration from anther cultures of seven rice varieties induced on N6 basal medium plus 2 mg' L -1 NAA and 0.5 mg' L -1 KIN in Protocol 1 calli were transferred from dark to light at 30 d without subcultures. In protocol 2, calli were subcultured to MS plus 0.1 mg ' L-1 NAA and 1 mg ' L- 1 BAP, prior to transfer to the light. Variety Yerua P. A. L. P. !tape P. A. Guayquirar6 Lemmont Sequial Basmati-370 Chajari

Anthers cultured

(%)

1072 743 750 648 409 616 407

13.0 20.0 15.0 13.5 15.0 25.0 9.0

callus

callus with green shoots (%) Protocol 1

Protocol 2

80.75 62.2 70.0 71.2 46.8 30.0 63.0

29.7 21.8 40.5 19.4 0.00 21.6 38.0

vesi (1982), who reported a frequency of diploid plants of 60 %. Plants transferred to soil flowered, and nearly 75 % of these produced seeds. This suggests that chromosome duplication occurred later or that the plants were chimeric (Fig. 3 b).

614

M. A. MARASSI, O. A. Bovo, G. L. LAVlA, and L. A. MROGINSKl

Acknowledgements

The authors are grateful to Mr. H. Vazquez for preparing the illustration, and Ing. Agr. Juan Justo N . Marassi, Estacion Experimental Julio Hirschhorn, Facultad de Agronomia, Universidad Nacional de La Plata and Ing. Agr. Rodolfo Vicino, Centro Operativo Colonia Masdas, Ministerio de Agricultura, Provincia de Santa Fe, Argentina for supplying the plant materials used in this work.

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