Bot. Bull. Acad. Sin. (2002) 43: 139-146

Chi — Various embryo rescue methods in interspecific crosses of lily

The efficiencies of various embryo rescue methods in interspecific crosses of Lilium

Hai-Shan Chi

Department of Horticulture, National Chiayi University, Chiayi 600, Taiwan, Republic of China

(Received July 16, 2001; Accepted January 22, 2002)

Abstract. The purpose of this research was to compare the efficiencies of various embryo rescue methods in interspecific hybridization of lily. This research included ten combinations of crosses with six crossing types (Lilium longiflorum X Asiatic hybrid, LA; Oriental hybrid X Asiatic hybrid, OA; Oriental hybrid X Lilium longiflorum, OL; Lilium longiflorum X Oriental hybrid, LO; Asiatic hybrid X Oriental hybrid, AO; and Asiatic hybrid X Lilium longiflorum, AL). Four culture methods—ovary slice culture (OSC), ovule with placenta culture (OPC), young single ovule culture (YOC), and embryo (sac) rescue method (ESR)—were used to culture the fertilized ovules after cut style pollination on plants. The OSC method could be used to obtain hybrid plants in each of the crossing types (0.1-2% success), except for in the OA, AO, and AL crossings. With the OPC method, ovule germinated (0.2-0.8% success) only when Lilium longiflorum "Gerlia" was used as the maternal parent. OSC and OPC required more complicated manipulations. When using the YOC method, ovules were harvested 10 days after the interspecific crosses, and embryo could be germinated from various genotype combinations (0.1-1.1% success). The number of days after pollination (DAP) required for ovule germination in this method (60-125 DAP) was fewer than in the OSC (73-155 DAP) and OPC (57-152 DAP) methods. More plantlets were obtained by the ESR method. The value of the ESR method is that, in the OA crossing, the embryo could only be germinated (0.2-0.3% success) after about 2-3 months of cut style pollination. Apomixes were found in the ovules in some of the cases in this crossing type. No plantlets were obtained in the AL or AO crossings with any of the culture methods tested. Based on the data obtained here, the ESR appears to be the most effective method for obtaining hybrid plants in interspecific hybridization of lily.

Keywords: Apomictic embryo; Cut style pollination; Embryo (sac) culture; In vitro culture; Interspecies crosses; Lilium; Ovary slice culture; Ovule with placenta culture; Young single ovule culture.

Abbreviations: A, Asiatic hybrid; O, Oriental hybrid; L, Lilium longiflorum; AP, Oriental hybrid "Acapulco"; CB, Oriental hybrid "Casa Blanca"; CK, Asiatic hybrids "Connecticut King"; MB, Asiatic hybrids "Mont Blanc"; G, Lilium longiflorum "Gelria"; IS, Lilium longiflorum "Indian Summer"; SQ, Lilium longiflorum "Snow Queen"; DAP, days after pollination; E (sac), embryo and/or embryo sac; CSM, cut style pollination method; OSC, ovary slice culture; OPC, ovule with placenta culture; YOC, young single ovule culture; ESR, embryo (sac) rescue method.

Introduction

In the genus Lilium, interspecific hybridization has been conducted to produce novel hybrids which can combine the Botrytis resistance of Orientals, the virus resistance of Asiatics, and the Fusarium resistance of trumpets with attractive blooms and good growing qualities. Various techniques have been applied successfully to overcome pre- and post-fertilization of incompatibility. A comparison of various pollination methods concluded that pre-fertilization barriers could be overcome by using the cut style technique (Van Tuyl et al., 1982, 1988). This method applies the pollen on the stylar surface after removing the stigma and part of the style, which inhibit pollen tube growth. Once fertilization has occurred, the growth of the hybrid embryos may still be restricted by post-fertilization barriers. These may be partly overcome by using the embryo rescue method in lily as described by several authors (North and Wills, 1969; Asano and Myodo, 1977; Asano, 1980; Van Tuyl et al., 1986).

While post-fertilization barriers may be overcome by placenta culture (Janson, 1993; Niimi et al., 1995) and ovary slices may be cultured successfully starting 40 days (Kanoh et al., 1988) or 5-8 days after pollination (Van Tuyl et al., 1991), the integrated use of cut-style pollination followed by embryo rescue has generated some new interspecific hybrids (Van Tuyl et al., 1991). A drawback associated with the cut-style method, however, is the low seed set (Van Tuyl et al., 1982, 1986), presumably caused by the premature arrival of pollen tubes in the ovary (Janson et al., 1993) and the low rate of pollen tube penetration into the micropyle of ovules instead of sperm cells formation (Chi, 2000). Van Tuyl et al. (1991) suggested that the low number of hybrid plants recovered might be attributable to the relatively long interval between pollination and embryo rescue (Van Tuyl et al., 1991). A simple method for obtaining seedlings from very young embryos was established in L. formosamum by the young single ovule culture (YOC) technique (Niimi et al., 1995). Niimi et al. (1995) found that seedlings could be obtained from

Botanical Bulletin of Academia Sinica, Vol. 43, 2002

Four rescue methods were used. The ovary slice culture (OSC), young ovule culture (YOC), and ovule with placenta culture (OPC) methods were initiated 10-11 DAP, and the embryo (sac) rescue (ESR) method was started 38-71 DAP, depending on the crossing type and combination. The culture media are given in Table 2.

Ovary slice culture. The ovary was transversely sectioned 10 DAP into six to eight slices of 3-4 mm thickness and polarly placed on the ovary slice culture medium (Table 2) followed by ovule culture 40-86 DAP. The ovules were excised one by one from the ovary slice disks and cultured on ovule culture medium until germination (Table 2).

Ovule with placenta culture. Ovules with placenta were excised from ovaries and incubated on MS medium (Table 2), the ovules were excised individually 40-71 DAP from the placenta and cultured on ovule culture medium until germination (Table 2).

Young single ovule culture. Ten DAP, young ovules were excised from ovaries without placenta and ovary tissue and incubated on MS medium until germination (Table 2).

Embryo/embryo sac culture method (E/Esac). Embryo (sac) culture was applied 38-70 DAP, depending upon the crossing type. Embryos and/or embryo sacs were removed from the ovules under a dissecting microscope and cultured on the embryo culture medium until germination, depending on genotypes and combinations (Table 2).

Following the rescue procedure, all of the materials were incubated at 25°C in the dark. The number of ovules germinating were recorded daily for six months.

embryos 10 days after self-pollination by culturing the single ovules, although the success rate was relatively low (below 5%). This method may be used for producing novel cultivars from crosses between distantly related species in which death of hybrid embryos occurs at a very early stage (Niimi et al., 1995).

In this report, young embryos (10 days after pollination) of Lilium interspecific crosses were used to determine the efficiency of embryo germination among four embryo rescue techniques.

Materials and Methods

Plant Materials

Seven cultivars from three groups, namely Oriental hybrids (O): "Acapulco" (AP) and "Casa Blanca" (CB); Asiatic hybrids (A): "Connecticut. King" (CK) and "Mont Blanc" (MB), and Lilium longiflorum (L): "Gelria" (G), "Indian Summer" (IS), and "Snow Queen" (SQ) were used to perform interspecific crosses in a phytotron (26°C). Before pollination, the plant materials were grown in a greenhouse with temperatures varying from 15°C at night to 20-25°C during the day with summer peaks of 30-35°C. There were a total of six interspecific crossing types (AO, OA, OL, LO, AL, LA) and ten combinations of crosses (Table 1).

Methods

The cut style method (CSM) was used for interspecific crossing (Van Tuyl et al., 1991). Ovaries were harvested 10-11 days after pollination and were surface-disinfected with 70% ethanol for 1 min and then with a commercial bleach solution containing 1.8% chlorine for 10 min, followed by three rinses with sterilized distilled water.

Chi — Various embryo rescue methods in interspecific crosses of lily

Results and Discussion

Ovary Slice Culture (OSC)

The highest ovule germination was obtained with the LA crossing type. The number of ovules germinated was higher in Lilium longiflorum "Gerlia" X Asiatic hybrid "Montblanc" (G X MB) (19 ovules germinated; 2%) than in Lilium longiflorum "Gerlia" X Asiatic hybrid "Connecticut king" (G X CK) (10 ovules germinated; 1%). The second place was the OL crossing type. Two (0.2%) ovules germinated in the Oriental hybrid "Acapulco" X Lilium longiflorum "Snow Queen" (AP X SQ) whereas only one (0.1%) germinated in the Oriental hybrid "Acapulco" X Lilium longiflorum "Indian Summer" (AP x IS). The third was the LO crossing type; only one ovule germinated (0.1%) in Lilium longiflorum "Indian Summer" X Oriental hybrid "Acapulco" (IS x AP) (Table 1).

Fernandez et al. (1996) suggested that the initiation time of ovary slice culture greatly influenced the recovery of seedlings. Ovary slice culture can enhance the chance of success in developing new hybrids if initiated at least 10 DAP (Fernandez et al., 1996). Kanoh et al. (1988) suggested that the ovary wall or placenta tissue in the MS medium may supply nutrients or hormones not available

in the culture medium but would stimulate hybrid embryo germination (Kanoh et al., 1988). In these experiments, almost all of the crossing types involved could be used in this method (except for the OA, AO, and AL) to obtain plantlets even though the efficiency (the range of percentage of ovule germination was about 0.1-2%) was not high in comparison with the large number of ovules (8821) cultured. However, transferring the ovules individually from the ovary slice to the ovule culture medium is time-consuming. In some cases, the ovules germinated directly on the ovary slice (Figure 1). In the OA, AO, and AL crossing types, no germination occurred.

The amount of time requiring for ovule germination in this culture method was 73-155 DAP, depending on crossing type and genotype used.

Ovary with Placenta Culture (OPC)

The highest ovule germination was obtained with the LA crossing type. The number of ovules germinated (3; 0.4%) in Lilium longiflorum "Gerlia" X Asiatic hybrid "Montblanc" (G x MB), was the same (3; 0.3%) as in Lilium longiflorum "Gerlia" X Asiatic hybrid "Connecticut King" (G x CK). In the LO crossing type, only one ovule germinated (0.2%) in Lilium longiflorum X Oriental hybrid

Botanical Bulletin of Academia Sinica, Vol. 43, 2002

"Acapulco" (G x AP) (Table 3). In this method, the hybrid embryo was able to germinate only when the mother plant was Lilium longiflorum "Gerlia."

Niimi et al. (1995) found that the ovule with placenta culture method seems to be more practical than the ovule and ovary slice culture method for rescuing young embryos 10 days after self-pollination in L. formosanum Wallace. They suggested that this method might be applicable for producing novel cultivars from crosses between distantly related species (Niimi et al., 1995). In this study, the range of ovule germination was 0.2-0.4% with this method. However, its efficiency (0.2-0.8%) was lower than that of the ovary slice culture method (0.1-2%), and it was also time-consuming to transfer the ovules from the placenta to the ovule culture medium. In some cases, the ovules germinated directly on the placenta (Figure 2). In the OA, AO, and AL crossing types, no ovules germinated.

The amount of time requiring for ovules germination in this culture method was 60-152 DAP, depending on the crossing type and genotype used.

Young Single Ovule Culture (YOC)

The highest ovule germination was obtained in the LA crossing type. The number of ovules germinated (8; 1.1 %) was higher in Lilium longiflorum "Gerlia" X Asiatic hybrid "Connecticut King" (G x CK) than in Lilium longiflorum "Gerlia X Asiatic hybrid "Montblanc" (G x MB) (4; 0.7%). Only one ovule germinated in the Oriental hybrid "Acapulco" X Lilium longiflorum "Snow Queen" (AP X SQ; OL crossing type; 0.1%) (Figure 3) or Lilium longiflorum "Gelria" X Oriental hybrid "Acapulco" (G x AP; LO crossing type; 0.2%) (Table 4).

In L. pumilum, the endosperm nucleus divides by mitosis into two nuclei 8-14 days after pollination. At this time, the embryo is found to be a ball of 4-8 cells. The endosperm is the tissue that nourishes the young embryo from fertilization to the time when the green leaves become functional. During the next 14-21 days, the endosperm grows rapidly (Brandram and Dowrick, 1969). For crosses between distantly related species death of hybrid embryos often occurs at a very early stage. Brandram and Dowrick (1969) found that in the cross, L. taliense × L. lankongense, abnormalities were seen in endosperms 12-13 days after pollination. After 14 days, many nuclei became degenerated. Abnormalities (most often two or three nuclei joining together) were observed in some ovules at varying periods up to 18 days after pollination. A well developing embryo was associated with a degenerate endosperm in some samples (Brandram and Dowrick, 1969). Therefore, the young ovule culture could be started at 10 days after interspecific crosses and embryo could be germinated (Figure 3). The range of ovule germination in this method was 0.1-1.1%. In this method, one more crossing type (LA, LO, and OL) germinated ovules than in the ovule with the placenta culture method (LA and LO). And in the LA crossing type, more ovules germinated (0.7-1.1%) than in the ovule with the placenta culture method (0.7-0.8%). The total time required to reach ovule germination in the single ovule culture method (60-125 DAP) was less than in the ovule with the placenta culture (57-152 DAP) and the ovary slice culture (73-155 DAP) methods. The single ovule culture method (0.1-1.1%) had a higher percentage of ovule germination than the ovule with the placenta culture method (0.2-0.4%) but a lower percentage than the ovary slice culture (0.1-2%). In the OA, AO, and AL crossing types, no ovules germinated.

Chi — Various embryo rescue methods in interspecific crosses of lily

The Embryo (sac) Culture (ESR)

Although the actual rate of fertilization may be higher because some embryos died before being rescued, the number of rescued embryos can be used to estimate the minimum rate of fertilization. In the embryo rescue method, the product of this rate of fertilization and the percentage of germination is defined as the real percentage of embryo germination (Table 5). Crossing type OA has the highest rate of fertilization (3%) followed by the LA (1%), OL (AP x SQ, 0.4%), and LO (IS x AP, 0.3%). In almost all crossing types (except the AO and AL crossing types), embryos germinated in this embryo rescue method. In the LA crossing type, most of the embryos were cultured 38 DAP, and 26-34% embryo germination was obtained 67-82 DAP. The number of ovules germinated was higher in Lilium longiflorum "Gelria" X Asiatic hybrid "Montblanc" (G x MB) (38; 34%) than in Lilium longiflorum "Gelria" X Asiatic hybrid "Connecticut King" (G x CK) (20; 26%). In the OL crossing type, the embryo sacs were cultured 38 DAP, but only three embryos germinated (14%) in the Oriental hybrid "Acapulco" X. Lilium longiflorum "Snow Queen" (AP x SQ) 119 DAP. In the LO crossing type, two embryo sacs were cultured 40 DAP, and only one embryo germinated in Lilium longiflorum "Indian Summer" X the Oriental hybrid "Acapulco" (IS x AP) 98 DAP. In the OA crossing type, the embryo sac was rescued starting 64-71 DAP, 8-10% embryo germination was obtained 92-128

DAP. The percentage of embryos germinated in the Oriental hybrid "Casablanca" X Asiatic hybrid "Connecticut King" (CB x CK, 4, 10%) was higher than in the Oriental hybrid "Casablanca" X Asiatic hybrid "Montblanc" (CB x MB, 14, 8%) (Table 5). No embryos germinated in the AO or AL crossing types.

In the OA hybridization, the embryo, which grew fairly well in the embryo sac without the endosperm (Figures 4-6) was rescued and after that embryo germinated fairly well under in vitro conditions (Figure 7). It seemed that embryo development stopped during the 2-3 months of the embryo growing period by post fertilization barriers, probably due to the lack of endosperm when embryos of OA developed under in vivo conditions (Figures 4-6).

Among these four embryo rescue techniques, the actual percentage of ovule germination in the embryo rescue method was the lowest. The value of the embryo rescue method is that in OA crossing only the embryo rescue method resulted in embryo germination. The time needed to germinate embryos after pollination was shorter than in other culture methods in the LA crossing type.

Apomictic Embryos Formation in the Genus Lilium

In the OA hybridization ("Casablanca" x "Montblanc"), twin embryos were obtained in the same embryo sac (Figure 8), but they couldn't germinate afterwards. This is

Botanical Bulletin of Academia Sinica, Vol. 43, 2002

Chi — Various embryo rescue methods in interspecific crosses of lily

consistent with the finding of Myodo (1975) that, in the cross of L. formosanum × L. leichtlinii var. maximowiczii, a group of cells in the nucellor tissue began to form an apomictic budding. The location of the cells that formed an apomictic embryo was not constant, but in most cases it was in the vicinity of the micropyle. The frequency of their location in the nucellar tissue was 87% of all the observed apomictic embryos initiated in the nearest portion to the micropyle (Myodo, 1975). Nogler (1984) also observed that aposporous initials develop in a zone adjacent to the progenitor cells of a sexual megagametophyte.

In applying these four embryo rescue techniques, it is important to determine the optimal timing for each method and cross. For lily, the abortion of the embryo is often caused by the degeneration of the endosperm or absence of the endosperm (Asano and Myodo, 1977). Therefore, embryo rescue methods (ESR) have to be applied. However, between fertilization and the start of embryo culture, a lot of embryos are already lost. Employing OSC, OPC, or YOC, it is possible to rescue embryos in an earlier phase. However, in interspecific crosses using Asiatic hybrid lilies as the female parent, no embryo germination was obtained in any of the four methods. This may give certain combinations of genes that are either not viable themselves or which are incompatible with the embryo or maternal tissues.

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Botanical Bulletin of Academia Sinica, Vol. 43, 2002