WO1990010383A1

WO1990010383A1 - Asexual propagation of genetic-cytoplasmic male sterile and/or other onion lines

Info

Publication number: WO1990010383A1
Application number: PCT/US1990/001325
Authority: WO
Inventors: Leonard M. Pike; Kil S. Yoo
Original assignee: The Texas A&M University System
Priority date: 1989-03-13
Filing date: 1990-03-12
Publication date: 1990-09-20
Also published as: AU5286990A; IL93717A0; NZ232876A; ZA901877B

Abstract

A tissue culture process for the asexual propagation of plants of the genus Allium is described and claimed. The method involves culturing, on first and second culture media, immature flower buds obtained from umbels of the plants prior to opening of the umbel spathes. The method provides many more plants than other methods of asexual propagation and permits use in hybrid seed production of genetic-cytoplasmic male sterile plants for which no genetically similar maintainer lines are available.

Description

ASEXUAL PROPAGATION OF GENETIC-CYTOPLASMIC MALE STERILE AND/OR OTHER ONION LINES

Onions, together with related species, including garlic, leek, and chives, are an important horticultural crop. The onion has been greatly improved in characteristics such as quality, yield, uniformity, pungency, and flavor through breeding programs designed to produce cultivars that suit prevailing demand. In the past, most cultivars or varieties have been bred using conventional techniques for the production of hybrid seed, such as backcrossing. In general, two parent plants with different traits are crossed to produce an Fl generation, followed by subsequent generations of inbreeding with the aim of obtaining new combinations of traits. After inbreeding for a number of generations, a line is produced which is relatively uniform and distinct for a number of traits or characters and which can be distinguished from other lines or cultivars. Following evaluation, a new line may be released as a new cultivar.

Pollination control in a cross-pollinating species such as onions is extremely difficult due to the floral biology of the plants. When onion plants are induced to flower, each plant produces one or more indeterminate inflorescences called umbels. Each umbel may contain a few to more than 2,000 flowers, typically 300 to 400 flowers. The normal flower in onions is perfect, but due to delayed female maturity, cross pollination is favored. To make a cross between two lines, a breeder has several choices. One is to hand-emasculate the stamens from one of the lines. This is an extremely difficult, time consuming and costly procedure. Another procedure is to use a genetic-cytoplasmic male sterile line. Male sterile plants produce normal flowers except that the pollen does not develop into a viable stage.

To utilize the male sterile condition in hybrid development, the breeder must develop pairs of breeding lines known as A and B lines. The A-line, known as the female line, is male sterile, but has normal female fertility. The B-line, known as the male or maintainer line, has the same nuclear genotype as the A-line, but has a different cytoplasm (normal or fertile cytoplasm) . Male sterility is conferred by the interaction of a recessive nuclear gene and a cytoplasmic factor. The cytoplasmic factor is designated N for normal fertile cytoplasm and S for the sterile condition. The nuclear genetic condition is designated Ms/_ for the normal fertile condition and ms/ms for the sterile condition. Table 1 illustrates why male sterility can only be maintained by having two lines with the following condition: the female line must be homozygous for Sms/ms and the male line must be homozygous for the Nms/ms.

TABLE 1 Progenies Resulting from Various Genetic and

Cytoplasmic Combinations Crossed onto a Male Sterile Onion Line⁸

Male sterile line (A) Male fertile line (B). Progenies →Nms/ms →Sms/ms

Sms/ s →NMs/ms →SMs/ms →Sms/ms

→NMs/Ms →SMs/ms →SMs/Ms →SMs/ms →SMs/ms →SMs/ms →Sms/ms

^aOnly the Sms/ms condition is male sterile.

The fertile cytoplasmic factor does not transfer to the female during the cross. Therefore, the N cytoplasm provides for normal fertility in the maintainer, yet does not restore fertility in the progeny when crossed onto the male sterile line. Any other combination of genetic and cytoplasmic factors in the maintainer line will give either fertile or segregating progenies. Thus, male sterile seed parents are maintained and increased using genetically similar maintainer lines having the genetic sterile-cytoplasmic fertile condition. However, many male sterile plants occur in onion progenesis within breeding lines and open pollinated cultivars which previously could not be economically utilized because no genetically similar maintainer line could be found.

The concept of the present invention followed several failures to find a maintainer line for segregating male sterile plants in the TAES developed cultivar Texas Grano 1015Y (Plant Variety Protection Certificate No. 8200170) . The idea of using asexual propagation methods dates back several years. One reported method involved inducing bulbil formation in the umbel. The method included the steps of trimming away and discarding the immature flowers and then ^{. .}

— 4 —

inducing bulbils or small plants to form in the umbel with the aid of water or chemical sprays. See Andrews, W. T. (1951) , "Vegetative reproduction of onions by the headset method," J. Amer. Soc. Hort. Sci. 58:208-212, and Thomas, T. N. (1972) , "Stimulation of onion bulblet production by N⁶-Benzyladenine," Hort. Res. 12:77-79. Another method was directed to the culture of onion flower heads or umbels, in various stages of micro- sporogenesis. The umbels were divided into halves, quarters, eighths or sixteenths and the sections were cultured. The method produced an average of, at best, a hundred shoots per capitulum, or flower head. Dunstan, D. I. and Short, K. C. (1978) , "Shoot production from the Flower Head of Allium cepa L.," Scientia Hort. 10:345-356.

Most recently, a method was reported at the 4th Eucarpia Allium Symposium which was directed to asexual propagation of winter leeks. The abstract of the Symposium presentation referred generally to culture of buds, flowers, or flower parts, but gave no indication of the stage of umbel development and no details of the culture method. See Baumunk-Wende, E. (1988) , "Application of tissue culture in hybridization of Allium porrum L. (Abstract)," Eucarpia, Proceedings of the 4th Allium Symposium, p. 99.

In contrast to these procedures, the present invention requires removal of the umbel prior to the opening of the umbel sheath, or spathe. Individual flower buds are then removed from the umbel and cultured into plants. According to the present invention, a thousand or more plants can be cultured from a single desirable donor cultivar.

Thus the present invention provides a method for rapidly propagating male sterile plant lines without using maintainers. It also produces many more plants than the closest known similar method of asexual production.

It is, therefore, an object of the present invention to provide a process for the asexual propagation of plants of the genus Allium that will permit the efficient and rapid production of a large number of individual plants from a single desirable plant cultivar without resort to long term back crossing which requires eight to ten years.

It is also an object of the present invention to provide a process for the asexual propagation of plants of the genus Allium that will permit the utilization of genetic-cytoplasmic male sterile plants which have no maintainers in hybrid seed production.

In accomplishing the foregoing objects, there has been provided, in accordance with one embodiment of the present invention, a tissue culture process for the asexual propagation of onions, which are members of the species Allium cepa L. In the process described, a thousand or more individual plants may be rapidly produced from a single onion cultivar. The process comprises the following steps. Using normal production practices, onion plants of the desired cultivar, e.g. genetic-cytoplasmic male sterile onion plants, are brought into flowering stage. Each plant produces umbels containing immature flower buds. Prior to the opening of the umbel spathes, the complete umbels are removed from the plants and sterilized. The umbel spathes are then removed and the individual flower buds or immature flowers are placed on a first culture medium under conditions of temperature and light appropriate to promote shoot formation and plant growth. When the shoots reach 2 to 3 cm in length, they are transferred to a second culture medium under conditions of temperature and light appropriate to promote root formation and plant growth. After roots have formed and the plants have been hardened, the plants are transplanted into growing trays filled with a soil mix to promote further growth. The young plants are removed from the growing trays and planted in the field during the fall. They are allowed to grow over the winter so that they will flower the following spring. Male fertile onions are also planted to provide pollen for production of hybrid seed. The following spring, the onion plants flower and pollination occurs, resulting in hybrid seed production.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, the detailed description and specific example is given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

FIG. 1 is a series of schematic representations, labelled horizontally as Stages (a) , (b) and (c) and numbered as Steps l through 10, depicting one embodiment of the present invention, the asexual propagation of genetic-cytoplasmic male sterile onions and their use in production of hybrid seeds.

FIG. 2 illustrates placement of immature flower buds on a first culture medium. FIGS. 3 and 4 illustrate formation of multiple shoots on individual flower buds.

FIG. 5 illustrates transfer of the shoots to a second culture medium.

FIG. 6 illustrates transplantation of the young plants to a soil mix following root development.

The present invention relates to processes for the asexual propagation of plants of the genus Allium. wherein many new plants may be grown that exactly duplicate the characteristics of a single desirable plant cultivar. The present process was developed for Allium cepa L. or onions, but may also be appropriate for plants having characteristics similar thereto, such as Allium ampeloprasum L. (great-headed garlic, leek, and kurrat) , Allium chinense G. Don (rakkyo) , Allium fistulosu L. (Japanese bunching or Welsh onion) , Allium sativu L. (garlic) , Allium schoenoorasum L. (chives) , and Allium tuberosum Rottler ex Sprengel (Chinese chives) . In addition, the method has been applied with similar success to broccoli and cauliflower.

As illustrated in the schematic diagram of FIG. 1, one embodiment of the present invention involves culturing the immature flower buds of genetic- cytoplasmic male sterile onion plants for use in hybrid seed production.

Stage A of FIG. 1 depicts the steps involved in obtaining the immature flower buds from the donor plants. In Step 1 of Stage A, the genetic-cytoplasmic male sterile onion plants are grown to flowering stage. In Step 2, the umbels, including 1 cm of flower stem, are cut from the donor plants, just prior to opening of the spathes which encase the immature flower buds. The umbels are then taken to the tissue culture laboratory and immersed in a sterilizing solution. A rinse of sterilized distilled water is unnecessary because, in Step 3, the sterilized umbels are cut into halves longitudinally and the spathes and flower stem tissues are discarded. The immature flower buds inside the closed spathes are usually free from contamination. In Step 4, the immature flower buds contained in each umbel are removed, under sterile conditions, along with 2 to 3 mm long stalks, or pedicels. The length of the pedicels is not important, but the shorter pedicels are easier to handle.

Stage B of FIG. 1 depicts the steps involved in culturing the immature flower buds. In Step 5 of Stage B, the flower buds are placed in tissue culture containers on a first culture medium which promotes shoot development. Three to five buds can be placed in a culture tube, or, as illustrated in FIG. 2, approximately 20 buds can be placed in a petri dish. Placing the flower buds upright is preferred but the direction is not critical. The culture tubes or dishes are then covered and placed under conditions of temperature and light appropriate to initiate shoot formation from the flower buds. Shoot formation on onion flower buds, which is illustrated in FIG. 3, typically occurs 4 to 6 weeks after placement on the first culture medium. The number of shoots formed on each onion flower bud averages about 5, and ranges from 2 to 10, as illustrated in FIG. 4. When the shoots have attained a length of about 2 to 3 cm, they are transferred, as shown in Step 6 of FIG. 1 and in FIG. 5, to culture tubes or dishes containing a second culture medium which promotes root formation. Again, the culture tubes or dishes are covered and placed under conditions of temperature and light appropriate to stimulate plant growth. After roots have formed, the covers of the tissue culture containers are gradually lifted to reduce the relative humidity in the containers and to harden the plants. As plant growth occurs, the covers are completely removed. Typically, in 4 to 5 weeks from transfer to the second culture medium, the young onion plants are ready for transplanting to the greenhouse. In Step 7 of FIG. 1, and as shown in FIG. 6, the young onion plants are transplanted to growing trays filled with a soil mix to allow further growth. The young plants are allowed to grow until the two to three leaf stage.

Stage C of FIG. 1 depicts the final steps of hybrid seed production. In Step 8 of FIG. 1, the young plants are removed from the growing trays and planted in the field during the fall. They are allowed to grow over the winter so they will flower the following spring. Male fertile plants are also planted to provide pollen for production of hybrid seed. In Step 9, which occurs the following spring, the onion plants flower and pollination occurs. Finally, in Step 10, hybrid seed production is complete and the seed is ready for harvest.

As is apparent from the foregoing description, the present invention provides a rapid and efficient means to produce large numbers of genetically identical plants from the flower buds of a single desirable plant cultivar rather than from seed.

In onions, each plant produces 3 to 5 umbels, each containing 300 to 400 immature flower buds. An average of 5 shoots are formed on each flower bud during culturing. Thus, a thousand or more plants can be obtained from the donor plant in a single growing season. Onions are commonly grown from transplanted seedlings to produce commercial and home garden crops. Therefore, handling asexually produced plants from a tissue culture method such as the present invention will fit well into established commercial practices. In addition, the method opens the way to produce hundreds of hybrids without having to have A and B lines (male steriles and their fertile maintainers) . With the ability to rapidly obtain large numbers of genetically identical plants from a single donor plant having desirable characteristics, hundreds of hybrids can be tested within a 5 year period where it once took up to 10 years to develop each male sterile line.

The following example presents an illustrative but non-limiting embodiment of the present invention.

EXAMPLE 1

Texas Grano 1015Y onion plants were identified as being male sterile in seed production fields by observing their first flowering umbels. Umbels, including 1 cm of flower stem, were cut from the source plants just prior to the opening of the spathe. The size of each individual flower bud was about 1 to 2 mm in diameter.

The flower heads were immersed in 70% alcohol for 1 minute, then immersed in a 10 vol % commercial bleach solution containing one or two drops per 1,000 ml. of solution of a suitable surfactant, e.g., a polysorbate such as Tween 20. The commercial bleach comprised 5.25 wt. % sodium hypochlorite. The sterilized flower heads were then cut into halves longitudinally and the individual flower buds were removed along with 2 to 3 mm long pedicels under sterile conditions. The flower buds were then placed into culture tubes containing a first culture medium. The first culture medium was prepared as follows. A half strength of Murashige and Skoog salts (i.e. 2151.765 mg of Murashige and Skoog salts per 1000 ml water) was mixed with 0.5 mg thia ine-HCl, 100 mg yo-inositol, 1.0 mg pyridoxine, 5.0 mg nicotinic acid, 30 g sucrose, 5.0 mg benzylaminopurine, and 0.5 mg naphtaleneacetic acid in 1,000 ml distilled water. The pH was adjusted to 6.0 and then 7 g of agar were mixed into the solution. An 8 ml volume of dissolved medium was then dispensed into vials (25 x 100 mm) and autoclaved at 125°C and 1.5 kg/cm² for 15 minutes (when petri dishes were used, the autoclaved medium was poured directly into sterile dishes) . Three to five flower buds were placed into each culture tube. The tubes were then covered with caps and placed in inflorescent light (100 uE/m² sec) under 12 hour daylength at 24°C.

After 4 to 6 weeks, shoot formation from the flower buds was initiated. When the shoots reached about 2 to 3cm long, the new plants were transferred to culture tubes containing a second culture medium, a sterilized vermiculite medium moisturized with half strength Murashige and Skoog salts. A volume of 1.5 solution of Murashige and Skoog salts to 1.0 volume of vermiculite was required to make an appropriately wetted mix because the vermiculite expanded after being autoclaved. The tubes were then covered with caps and place in inflorescent light (100 uE/m² sec) under 12 hour daylength at 24*1704*12*:*I704*10*C. After roots were formed, the caps on the tubes were gradually lifted. As plant growth occurred, the caps were completely removed. Usually it took 4 to 5 weeks from the transfer to the vermiculite medium for the plants to be ready for the next transplantation. The young plants were then transplanted into plant growing trays filled with a suitable soil mix such as Fison's Sunshine Mix, blend no. 1, to promote further growth and were kept in a greenhouse until they reached the two or three leaf stage.

The young plants were then ready to be removed from the greenhouse and planted in the field.

Claims

CLAIMS :

1. A tissue culture process for asexual propagation of plants of the genus Allium comprising:

cutting umbels having intact spathes from source plants that are approaching flowering;

sterilizing said umbels;

removing individual flower buds with pedicels from said sterilized umbels;.

placing said flower buds with pedicels on a first culture medium in covered tissue culture containers and culturing said flower buds under conditions of temperature and light appropriate to promote the formation of shoots;

transferring said shoots, when they have reached a length of 2 to 3 cm, to a second culture medium in covered tissue culture containers and culturing said shoots under conditions of temperature and light appropriate to promote root development and plant growth;

gradually lifting the covers of the tissue culture containers after roots have formed to reduce the humidity and harden the plants; and

transplanting said hardened plants into growing trays filled with a soil mix.

2. The method of claim 1 wherein said umbels are sterilized by immersing said umbels in a first solution comprising 70% alcohol and then immersing said umbels in a second solution comprising commercial bleach and a suitable surfactant.

3. The method of claim 2 wherein said umbels are immersed in 70% alcohol for a period of 1 minute.

4. The method of claim 2 wherein said second solution comprises 10% commercial bleach and one to two drops of a polysorbate surfactant per 1000 ml solution.

5. The method of claim 2 wherein said second solution comprises 10% commercial bleach containing as an active ingredient 5.25% sodium hypochlorite and one to two drops of Tween 20 per 1000 ml solution.

6. The method of claim 1 wherein the pedicels of said flower buds are 2 to 3 mm long.

7. The method of claim 1 wherein said first culture medium is prepared by

mixing a half strength of Murashige and Skoog salts, 0.5 mg thiamine-HCl, 100 mg yo- inositol, 1.0 mg pyridoxine, 5 mg nicotinic acid, 30 g sucrose, 5 mg benzylaminopurine and 0.5 mg naphtaleneacetic acid in 1000 ml distilled water;

adjusting the pH of the mixture obtained in step a) to 6.0; ixing 7 g of agar into the pH adjusted mixture obtained in step(b) ; and

autoclaving the mixture obtained in step (c) at 125°C and 1.5 kg/cm² for 15 minutes.

8. The method of claim 1 wherein said conditions of temperature and light appropriate to promote formation of shoots are inflorescent light (100 uE/m² sec) under 12 hour day length at 24°C.

9. The method of claim 1 wherein said second culture medium comprises sterilized vermiculite moistened with half strength Murashige and Skoog salts.

10. The method of claim 9 wherein the volume ratio of sterilized vermiculite to half strength Murashige and Skoog salts in said second culture medium is 1.5:1.0.

11. The method of claim 1 wherein the conditions of temperature and light appropriate to promote root development and plant growth are inflorescent light (100 uE/m² sec) under 12 hour day length at 24°C.

12. The method of claim 1 wherein said soil mix comprises Fison's Sunshine Mix, blend No. 1.

13. The method of claim 1 wherein the plants are genetic-cytoplasmic male sterile plants of the genus Allium-

14. The method of claim 13 wherein the plants are genetic-cytoplasmic male sterile plants of the genus Allium for which no maintainer lines are available.

15. The method of claim 1 wherein the plants are members of the species Allium cepa L.

16. The method of claim 15 wherein the plants are genetic-cytoplasmic male sterile plants of the species Allium cepa L.

17. The method of claim 16 wherein the plants are genetic-cytoplasmic male sterile plants of the species Allium cepa L. for which no maintainer lines are available.