PlantCeil Reports

Plant Cell Reports (1994) 13:637-640

9 Spfinger-Verlag1994

Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.) Cun-Jin Zhang ~, H o n g - L o n g Wang 2, , , Yun M a 2, and Yu-Qing Kang ~ 1 Department of Biology, Tianjin Institute of Education, Tianjin 300020, China 2 Department of Biology, Tianjin Normal University, Tianjin 300074, China * Present address: INRA, Symbiotes des Racines, 34060 Montpellier, France Received 11 September 1993/Revised version received 28 March 1994 - Communicated by A. Komamine

Abstract High percentages of micro-calli and micro-derived embryos were produced from isolated asparagus microspores at late uninucleate stage on MS liquid medium supplemented with 1.0 mg 1-1 2,4-D and 0.5 mg 1-1 BA. Two types of calli, namely compact callus (CC) and loose callus (LC), were found. Plantlets were regenerated via organogenesis, when these caUi were transferred onto MS solid medium supplemented with 1.0 mg 1"1 BA and 0.2 mg 1-~ IBA 6 weeks. Embryos were produced from liquid cultured microspores, or from solid cultured micro-calli. The frequencies of haploid plant production from organogenesis and embryogenesis were compared. Effects of plant growth regulators on callus production, plantlet regeneration, and haploid plant production were tested. The combination of BA 1.0 mg 1-1 and IBA 0.2 mg 1-1 resulted the highest precentage of haploid plant production (7.7% from CC, 4.3% from LC). Key w o r d s : Asparagus officinalis L. Microspore culture - Embryogenesis - Haploid - Plant growth regulators

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; IBA, 3-indolybutyric acid; BA, 6-binzyladinine; NAA, naphtalene acetic acid; MS, Murashige and Skoog. Introduction Isolated microspore culture provides an experimental system for gene transformation, mutant cell selection, haploid breeding, and study of embryo development at cellular, molecular and biochemical levels. Only a rather limited number of species were reported on the successful use of isolated microspore culture. Microspore-derived plants have been regenerated from isolated microspores of Correspondence to: H. L. Wang

wheat (Wei 1982), barly (Kohler et al. 1985), rice (Cho et al. 1988), maize (Pescitelli et al. 1990) and Brassica pp (Keller et al. 1987; Cao et al. 1990; Burnett et al. 1992). To our best knowledge, there are no report on asparagus haploid regeneration via isolated microspore culture. This paper deals with the production of haploid plants from isolated asparagus microspores. The critical factors include pretreatment of donor spikes and culture of microspores with different growth regulators. Frequencies of haploid plant production from organogenesis and embryogenesis were compared.

Materials and Methods Plant growth conditions. Plants of Asparagus officinalls L. (Mary Washington 500) were grown under greenhouse conditions(26+1~ supplemented light to extend daylength to 16 h). Lighting was provided by the cool, with fluorescent tubes. Plants were watered biweekly with fertilizer(100 ppm N, 50 ppm P, 120 ppm K). Isolation and culture of microspores. Flower buds 1.5-2,0 mm in length (containing microspores at late uniuncleate stage) were collected. Buds were placed in the dark at 6"C for 3 days, and then surface smrilized in 70% alcohol for 1 min followed by 3, 3-minute washes with sterile, distilled water. The buds were surface sterilized again in 0.1% HgCt2 for 8 min, and washed 3 times with sterile water. The anthers were stripped and placed in 100 ml of liquid medium contained in a 250 ml erelenmeyer flask. The erelenmeyer flasks were mantained at 28*(2 in the dark and agitated at 60 rpm. The anthers were taken out from the culture media after 3-5 days in cubation. The microspores released from anthers were continued to culture in flasks. Modified MS medium supplemented with inositol 5000 mg 1"1, glutamin 800 mg 1"1, serine 100 mg 1"1, and sucrose 0.3% were used. Various kinds of combinations of 2,4-D, BA, and NAA were supplemented to the media. The media were adjusted to pH 5.8 before sterilization.

638

Callus differentiation and plant regeneration. The mierospore-derived calli were transferred onto the MS solid media supplemented with casein hydrolysale 500 mg 1"1, inositol 500 mg 1"t, anti d'tfferent combinations of growth regulators. The 1-2 mm mierospore-derived embryos were transferred Ohm the MS solid medium containing IBA 0.3 mg

1"1. All solid ~ a . were adjusted to pH 5.8, 0.8% agar and 0.3% sucrose were ~_,~__,~:1before sterilization. The culture were incubated a t a temperature of 28~ under 12 h daily illumination with white fluorescent light(2,500 lx).

Cytological examinations. Plandet root tips (1.5 cm) were treated with 0.05 mg 1"1 colchicine for 1.5 h, then treated with 1N HC1 for 15 rain at 600C. After 10 min rinsing in alcohol, the materials were stained with acetocarmine, squashed gently and obeserved under the light microscopy. Results

and

Discussion

Microspore culture and callus formation The microspores were cultured on the MS liquid medium supplemented with 5000 mg 1-1 inositol, 800 mg 1-1 glutamine, 100 mg 1-1 serine, 3% sucrose. Various kinds of combinations of different growth regulators were examined in the microspore culture. The presence of 2,4D was seemly essential for the formation of microcalli (Table 1). Use of high concentration of 2,4-D up to 1.0 mg 1-1 in combination with 0.5 mg 1"1 NAA and 0.5 mg 1-1 BA resulted the highest frequency of micro-callus formation. But much higher 2,4-D concentration (1.5 or 2.0 mg 1-1)treatments decreased the frequency of microcallus formation. This is in the agreement with wheat microspore culture (Datta & Wenzel 1987). Low frequency of micro-caUi were produced, when the microspores were treated with 2,4-D alone. No micro-calli were yielded,

Increasing BA from 0.5 to 1.5 mg 1-1 in combination with 2,4-D 0.5 mg 1-1 and NAA 0.1 mg 1-1 stimulated the production of calli. But increasing BA from 0.5 mg 1-1 to 1.0 mg 1"1 in combination with 2,4-D 1.0 mg 1-1 and NAA 0.5 mg 1-1 resulted the decreasing the callus production.-: In addition, micro-callus formation was influenced by both stages of donor microspores and tempeture pretreatment of donor microspores (data not show). Microspores at late uninucleate stage were suitable for micros'pore division. Pretreatment of donor spikes at 6~ 3 days showed a significant effect on microspore division. Similar results were reported by other workers (Dore 1990; Wang et a1.1992; Zhang et al. 1992b). But in Brassica microspore culture, pretreatment of spikes with high tempetare (35~ was benefit to microspore division (Hamaoka et al. 1991; Baillie et al. 1992). The m i c r o ~ were cultured on the modified MS liquid medium supplemented with 1.0 mg 1-I 2,4-D, 0.5 mg 1"1 NAA, and 0.5 mg 1-1 BA. Large amount of microspores were obtained after 3-5 days incubation. In 4-day culture, the number of microspores reached the largest(50,000 ml~) in liquid media. Some microspores showed a roundedshape, with clearly visible cytoplasm and vacoues (Fig. A). These highly cytoplasmic microspores started to divide (Fig. B), and resulted numerous 4-cell microspores within 7-day incubation. Two types of microspore divisions were found. One is microspore equal division (Fig. C), the other is microspore unequal division (Fig. D). The majority of microspore divisions were unequal division. After 30 days in culture, the exines of the microspores were ruptured (Fig. E), then compact caUi (CC) (Fig. F), loose calli (LC) (Fig.G), and embryos (Fig. H, 13 were formed.

Plantlet regeneration Table 1. Effects of plant growth regulators callus induction from isolated microspores.

NAA (mg 1"1)

2,4-D

.

0.5 -0.5 0.5 0.5 0.5 1.0 1.0

1.5 2.0

.

.

.

0.1 0.1 0.1 0.1 0.1 0.5 0.5 0.5 0.5

BA

.

on

Frequencyof callus formation (%) 0

---0.5 1.0 1.5 0.5 1.0 0.5 0.5

5 0 10 12 20 25 5O 35

10 5

when asparagus microspores were cultured on the hormone-tree medium or treated with NAA alone.

Two types of micro-calli were transferred onto MS solid medium supplemented with 500 mg 1"1 casein hyrolysale, 500mg 1-1 inositol. Effects of growth regulators on organogensis and haploid plant production were tested (Table 2). Data in Table 2 showed that cytokinin and auxin take a significant effect on organogenesis and haploid production. Use of BA 1.0 mg 1-1 in combination with IBA 0.2 mg 1-1 resulted the highest precentage of plantlet regeneration (50.3% from CC, 33.7% from LC) and the highest frequency of haploid plant production (7.7% from CC, 4.3% from LC). No haploid plantlets were produced, when the calli were treated with BA alone, or BA 0.5 mg 1-I in combinations with IBA 0.2 mg 1"t or 0.5 mg 1-1.Increasing the concentrations of IBA (0.2-0.5 mg 1-1) in combination with BA 1.0 mg 1-1 resulted low frequencies of plantlet formation and haploid plant production, and high frequency of polyploid plant(_>3n) production. This means that higher concentration of IBA could induce ploidy variation of callus-derived plantlets.

639

F i g. A - B : Microspores with clear cytoplasm and the first division o f microspores(Bar= 100urn). F i g. C - I) : 4-cell microspores from equal and unequal microspore divisions(Bar= 100um). F i g . E : Microspore exine rupturing(Bar= 100urn). F i g. F : Compact callus(Bar=100urn). F i g . G: Loose callus(Bar=100um). F i g . H - I : Globular and other shaped embryos(Bar=100tma). F i g . J : Compact callus growing on MS solid medium. F i g . K : Embryos from callus. F i g . L : Plantlets from shoot initiation.

640

Fig.M: Plantlets from root initiation. plants in soil after 4-week transplanting;

F I g.N: Planflets from embryos,

Table 2. Effects of plant growth regulators on plantlet regeneration and haploid production. Callus

Growth No. of No. of No. of plantlets Percentage of regulators calli shoots ~ . . . . . . . . . . . . . . . . . . . . . . . . . differentiation BA + IBA cultured roots n 2n n-2n >3n (rag 1-1) initiated (%)

CC CC CC CC CC

0.5+0.2 0.5+0.5 1.0 + 0.2 1.0+0.5 1.0

412 506 348 436 408

44 76 112 87 36

12 5 3 40 8 12 12 10 10 7 2

2 6 3 9

14.1 17.8 50.3 29.4 11.0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

LC LC LC LC LC

0.5+0.2 0.5+0.5 1.0+0.2 1.0+0.5 1.0

318 401 416 503 450

22 53 94 75 32

1 24 15 -

3 4 16 7 5

1 2 6 4

6 3

8.5 14.7 33.7 20.1 9.8

Similar results were found in asparagus anther culture (Dore 1974; Zhang et al. 1992a, b). One possibility is that high concentration of IBA could induce amitosis of callus cells (Wang et al. 1991). Callus-derived plants contain a small amount of haploid plants and a large amount of polyploid plants, while the cytological test of embryo-derived plants showed that most of embryo-derived plants are haploids (data not show). This means that embryogenesis from isolated microspore culture provides an effective method for haploid plant production. Micro-calli were transferred onto modified MS solid medium supplemented with 1.0 mg1-1 BA and 0.2 mg1-1 IBA. All CC and most LC were grown fast (Fig. J), produced plantlets via shoot or root initiation (Fig. L, M) and embryos (Fig. K) within 2-week culture. Appromixately 20% LC only increased in mass after 8 subcultures. Plantlets were produced, when microsporederived-embryo and callus-derived-embryo were transferred to MS solid medium with IBA 0.2 mg 1q (Fig.

Fig. 0 : Haploid

(Zhang et al. 1992b). The frequency of plantlet regeneration from embryos reached 80%. But a few of embryos germinated, when they were transferred to hormone-free MS solid medium. This suggests that IBA takes a significant effect on the development of asparagus embryoids. This effect was also observed in asparaus anther cultures (Zhang et al. 1992a, b). A c k n o w l e d g e m e n t s . The authors wish to thank Dr J.D. Thompson for his revising the manuscript. References

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