WO2000016610A1

WO2000016610A1 - A process for the induction of direct in vitro organogenesis in onion

Info

Publication number: WO2000016610A1
Application number: PCT/SI1999/000022
Authority: WO
Inventors: Borut Bohanec; Zlata Luthar
Original assignee: Univerza V Ljubljani Biotehniska Fakulteta
Current assignee: Univerza V Ljubljani Biotehniska Fakulteta
Priority date: 1998-09-24
Filing date: 1999-09-22
Publication date: 2000-03-30
Anticipated expiration: 2001-03-24
Also published as: SI20053A; AU5768599A

Abstract

A process is described leading to the formation of direct in vitro organogenesis omitting the callus stage in onion, initiated from generative organs, particularly flowers or ovaries. Embryogenic tissues, formed in the process of regeneration are suitable for further attempts at genetic transformations. Direct somatic organogenesis is a method that provides a high degree of clonal propagation (micropropagation) starting from individual donor plants.

Description

A process for the induction of direct in vitro organogenesis in onion

Field of invention

This invention refers to a field of plant biotechnology, specifically to a new process for the induction of direct in vitro organogenesis in onion, with a specific application use in micropropagation or genetic transformation in onion.

Onion (Allium cepa L.) is the second most important vegetable species worldwide and is produced in almost all climatic regions. It can be multiplied by seeds, sets or vegetatively. Vegetative in vitro methods are used for multiplication of valuable breeding lines including maintaining male sterile lines, used in hybrid seed production. Methods of direct in vitro organogenesis can be further used for successful genetic transformation in onion. Prior Art

Publications concerning in vitro protocols in onion report of axillary and adventitious shoot proliferation of onion.

Micropropagation of onion presents several difficulties. Two different tissues have mainly been used for induction of shoot cultures - the first being inoculation of scale bases excised from the basal parts of bulbs or onion sets. This approach was taken in the following studies:

Hussey, G. (1978) In vitro propagation of the onion Allium cepa L. by axillary and adventitious shoot proliferation. Sci Hortic 9: 227-236;

Fujieda K., Matsuoka N., Fujita Y. (1979). Vegetative multiplication of onion (Allium cepa L.), through tissue culture. J Japan Soc Hort Sci 48: 186-194;

Hussey G., Falavigna A., (1980) Origin and production of in vitro adventitious shoots in the onion (Allium cepa L.). J. Exp. Bot. 31 : 1675-1686;

Kahane R., Rancollac M., Teyssendier de la Serve B. (1992) Long-term multiplication of onion (Allium cepa L.) by cyclic shoot regeneration in vitro. Plant Cell Tiss Org Cult 28:

281-288.

Another approach used immature flower buds as a starting tissue. Matsubara S., Hihara H. (1978) Onion bulblet regeneration on receptacles in vivo and in situ. J Japan Soc Hort Sci 46: 479-486 reports on the use of bases of immature inflorescences. Pike LM., Yoo KS. (1990) A tissue culture technique for clonal propagation of onion using immature flower buds. Sci Hortic 45: 31-36 and Mohamed-Yassen Y., Splittstoesser WE., Litz RE. (1993) In vitro bulb formation and plant recovery from onion inflorescences. Hort Science 28: 1052 used parts of immature inflorescences or individual immature flowers as explants. Callus tissues have been induced on a wider range of explant tissues, including bulb, set or seedling radicle (Dunstan DI. and Short KC. (1978) Shoot production from onion callus cultures. Sci. Hortic. 9: 99-110. Attempts have been made to induce embryogenic callus from seedling leaf sheets, immature sexual embryos, immature unfertilized ovules, mature basal plates (Phillips CG, Luteyn KJ (1983) Effects of picloram and other auxins on onion tissue cultures. J Amer Soc Hort Sci 108: 948-953).

The most frequently studied plant growth regulators (phytohormones) for shoot induction have been naphtaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), while for callus induction, picloram has been shown to be superior to NAA or 2,4- dichlorophenoxyacetic acid (2,4-D).

A characteristic of published protocols was a relatively low number of shoot formations per donor plant, protocols based on bulb scale parts resulting in up to 10 shoots per explant, while the explant number per one bulb was limited (Fujieda et al. 1979). Protocols based on immature flower buds resulted in about 10% induced flowers, which produced about 5 shoots per flower (Pike and Yoo 1990). Mohamed-Yassen et al. 1993 spliced immature inflorescences into 4 parts and achieved 10.6 shoots per explant making 42.4 shoots per inflorescence. None of the cited articles described a method of direct shoot organogenesis, which is the subject of this invention.

In vitro grown shoots can be subcultured according to Kahane et al. 1992, which is briefly based on swelling basal shoot parts, followed by splitting and further re-growth of micropropagated shoots. This multiplication cycle requires 3-4 months. On the other hand, the protocol published for somatic embryogenesis (Phillips and Luteyn 1983) required the induction of callus induced from meristem tips, seedling roots, mature embryos, immature fertilized ovules and parts of mature basal plates. Description of Invention with Working Examples

It is evident from the prior art references that an efficient method of direct in vitro organogenesis in onion, in which multiple shoot structures are induced on mature flowers or ovaries exclyding the callus stage, efficient on majority of tested cultivars, has not yet been developed. Such a method is advantageous for two reasons. Primary the formation of multiple shoot structures resulting in efficient vegetative multiplication of plants started from single individual plants. Secondary the use of direct somatic organogenesis as an important step in genetic transformation studies using a biolistic approach or via the Agrobacterium infection and similar methods.

We have now surprisingly found hat mature onion flowers or ovaries could be induced to form direct somatic organogenesis leading to the formation of multiple organogenic structures, which in later development form multiple shoots. The induction process and components of the media are very similar to those used for gynogenic embryo induction in onion (Bohanec B., Jakse M., Ihan A., Javornik B. (1995) Studies of gynogenesis in onion (Allium cepa L.): induction procedures and genetic analysis of regenerants. Plant Science 104: 215-224; Jakse M., Bohanec B., Ihan A.(1996). Effect of media components on the gynogenic regeneration of onion (Allium cepa L.) cultivars and analysis of regenerants. Plant Cell Rep 15: 934-938. The major differences between the protocol used for gynogenesis and the claimed process (protocol 2) are the use of BDS medium (Dunstan DL, Short KC. (1977) Improved growth of tissue cultures of onion (Allium cepa L.). Physiol Plant 41 : 70-72) instead of B5 induction medium (Gamborg OL., Miller RA., Ojima K. (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50: 151-158), elevated inositol and vitamin contents, the use of gellan-gum instead of agar solidified media and a shorter induction treatment. These relatively small differences cause a completely different regeneration response of the same onion organs. We believe that such responses have not been previously described.

The claimed invention is a process for induction of direct in vitro organogenesis in onion, comprising the steps of:

(i) Inoculation of flower buds at a mature stage but before dehiscence on induction media, which contain adequate concentrations of phytohormones, growth regulators and gelling agents for initiation of direct somatic organogenesis. Growth regulators at this stage include effective mixture of auxins and cytokinins, other media components include macro and micro elements, vitamins, inositol, proline, carbohydrates and gelling agents.

(ii) After an appropriate time of induction, transfer of explants from induction to differentiation media which contain adequate concentrations of cytokinin, macro and micro elements, vitamins, inositol, proline, sucrose and gelling agents, and growth until the occurrence of direct organogenesis.

(iii) Optional removal of perianth before the transfer from induction to differentiation media and culture of extracted ovaries. The following steps are the same as described in

(i) and (ii) leading to induction of direct somatic organogenesis.

(iv) Separation of globular embryogenic structures developed as described in (ii) and (iii) followed by transfer of elongated shoots to media for root development or maintenance of compact organogenic structures.

(v) Acclimatization of previously (iv) mentioned rooted shoots.

Characteristic features of the claimed invention are described in the following paragraphs, however the scope of invention is not limited therewith. The invention includes all possible variations which are obvious to the person skilled in the art. The applied differentiation and induction media contain as solidifiers gellan-gum, mixture of gellan-gum and agar, or only agar.

The duration of the growth on induction media is from 3 to 12 days.

The induction and differentiation media contain sucrose, glucose or maltose as a source of carbohydrates.

The induction and/or differentiation media contain 25-100 g/1 of sucrose.

In addition to cytokinin the induction media contain 2,4-dichlorophenoxyacetic acid or picloram as a sources of auxins.

The induction media contain auxin or auxin and 6-benzylaminopurine, thidiazuron or isopentenyladenin (2ip) as sources of cytokinins.

The differentiation media contain thidiazuron or 6-benzylaminopurine as sources of cytokinins.

The induction and differentiation steps are performed in light or in darkness.

The composition of macro and micro elements in induction and differentiation media corresponds to BDS media prepared according to Dunstan and Short, 1977, B5 media

(Gamborg et al. 1968) or MS media (Murashige and Skoog, 1962*).

*Murashige T., Skoog F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15: 473-497.

Detailed Description of the Process Embodiments

The plant material used in the following Examples originated from different, publicly available sources, cultivars were received from genebanks or were purchased at retail, and inbred lines were received from the US public breeding program (Dr. M.J. Havey, USDA, Madison, Wisconsin, USA). Various genotypes of onion were used in these experiments: Belokranjka (Slovenia), Ptujska rdeca (Slovenia), Stuttgarter Riesen, Timor, Shenshu Yellow, Yamaguchi Koudaka, Texas Early Grano 502, experimental hybrid XPH 3371 FI (Asgrow), hybrids 70723 (B1717BxB2923B) and 70719 (B2371CxB2923B), inbred lines B2355B, B2923B, MSU2935B, MSU5718B, MSU8155B.

Flower buds at a mature stage but before dehiscence were taken from above-referenced greenhouse grown donor plants and were surface sterilized by dipping for 10 min in 16.6 g/1 dichloroisocyanuric acid Na₂ salt with the addition of a few drops of Tween 20. Subsequent to this treatment the flowers were washed 3 times in sterilized water.

In embodiment 1 (of the claimed process), flower buds were cultured in Petri dishes 100 mm in diameter (30 per dish) on induction media described later. The flowers were on induction media for 6 days (where not said otherwise). After the induction period, flowers were subcultured on Petri dishes containing differentiation media, as described in the following paragraphs.

The embodiment 2 (of the claimed process) differed from embodiment 1 in that the flowers were cultured on induction media and were extracted (perianth removed) before transfer to differentiation media ovaries.

Petri dishes were sealed with Parafilm™ (American National Can, Greenwich, CT, USA) to prevent evaporation and exposed to a 16/8 hours photoperiod at 21-23 °C and illumination of approximately 80 μmol m^"2s^"'.

Media were prepared according to established protocols for plant tissue culture work, as described for example in RLM Pierik (1987) In vitro culture of higher plants. Martinus Nij ho ff Publishers, Dordrecht, Boston, Lancaster, p.344. The basal medium consisted of BDS macro, micro elements and vitamins (Dunstan and Short 1977, commercially available at Duchefa Biochemie BV Haarlem, Netherlands), 500 mg/1 inositol, 200 mg/1 proline, lOOg/1 sucrose (where not said otherwise), the pH was adjusted to 6.0 before autoclaving. Other media components for all exemplified treatments are listed in Tables 11 A and 1 IB.

Flower buds grown on induction media opened after the first few days, the ovaries enlarged significantly. Flowers and ovaries cultured on induction media and transferred to differentiation media formed the first visually perceptible structures after 3 weeks in culture. The formed structures were particularly notable in the second process embodiment (Protocol 2), where the perianth did not obstruct the view. At this stage, regenerated structures had a globular embryogenic appearance and were completely white (Fig. 1). Further development was evident within one week. The first visual shoot organogenesis appeared on globular structures in the following week (Fig. 2). A part of the shoot structures already elongated in the next 2 weeks on the differentiation medium, individual shoots being approximately 2 cm long (Fig. 3). Such shoots were divided and subcultured on the elongation media, on which they elongated and produced normal plantlets. A proportion of these organogenic structures remained as nodular bumps. When such clusters were subcultured on hormone free media, elongation of shoots occurred, although the organogenic potential was preserved for at least 3 subcultures. The most suitable medium for elongation of shoots was basal medium (or half strength basal medium) excluding phytohormones, with the concentration of sucrose or glucose lowered (20-70g/l, recommendable concentration 30g/l). Shoots gradually developed a green coloration, started to elongate and formed roots in the same way as shoots micropropagated using other standard methods. For accelerated root growth, the media could additionally contain auxins such as 0.5-1.0 mg/1 indolebutyric acid (IBA). The number of individual shoots per flower or ovary was difficult to determine because smaller compact organogenic structures were present in addition to elongated shoots. The average cluster at the end of subculture was composed of 5-10 elongated shoots, the rest being compact organogenic tissue.

The results presented in Tables 1-10 represent the number of flowers (first process embodiment »Protocol 1 «) or ovaries (Protocol 2) producing organogenic multiple shoot structures. Statistical differences determined separately for each genotype were tested with ANOVA followed by Duncan's multiple range test (p=0.05). Identical letters following values indicate no significant difference.

Example 1:

Effects of gelling agents

Media solidified using gellan gum (Il/Dl), agar/gellan gum mixture (I2/D2) or agar (Difco-Bacto™, Difco Laboratories, Detroit, MI USA) (I3/D3), were tested using Protocol 1 and Protocol 2. Flower buds were transferred from induction media to differentiation media after 6 days. Results are presented in Table 1.

Table 1:

The highest regeneration percentage of shoot differentiation occurred on media solidified with gellan-gum in both processes but more shoots exhibited a hyperhydrated appearance on gellan-gum solidified media than on agar or agar/gellan-gum mixture.

Example 2:

Duration of induction treatment

The varying duration of the induction stage was studied using the embodiment 2, Il/Dl media. The duration of the induction stage (3, 6 or 12 days) had a significant effect on regeneration. Results are presented in Table 2.

Table 2:

The duration of the induction stage had a significant effect on regeneration. The highest shoot regeneration was observed on ovaries that were placed on differentiation media after 6 days. Shorter or longer induction treatment resulted in decreased regeneration. At 6 days, the lowest hyperhydration also occurred.

Example 3

Effect of source of carbohydrates

To evaluate the optimal source of carbohydrates in induction and differentiation media, 50 g/l (I4/D4) sucrose was compared to an equimolar concentration of glucose (26.3 g/l - I6/D6) and maltose (52.6g/l - I7/D7). In this study. 3 genotypes and Protocol 2 were used. The results are presented in Table 3.

Table 3:

Organogenic structures occurred in all media and with all genotypes. There were no statistically significant differences among treatments, and a small difference in the occurrence of hyperhydration.

Example 4:

Effect of sucrose concentration in media

The influence of sucrose concentration in induction and differentiation media was studied using 3 genotypes and embodiment 2. Three different concentrations of sucrose used were: 100 g/l (Il/Dl), 50 g/l (I4/D4) and 25 g/l (I5/D5). The results are presented in Table 4.

Table 4:

Organogenic structures occurred in all media and with all genotypes. Medium supplemented with 50 g/l sucrose was significantly superior to 100 g/l and 25 g/l with genotype MSU5718B. In the other two genotypes, a lower content of sucrose resulted in a lower occurrence of hyperhydration.

Example 5:

Effect of auxin composition in induction media

The effect of auxin composition in induction media on 4 genotypes using the Protocol 2 was studied. Induction media contained 2 mg/1 2,4 D (14), 1 mg/1 picloram (18), 2 mg/1 picloram (19) or 5 mg/1 NAA (110). The differentiation media were Dl and D4. The results are presented in Table 5.

Table 5:

Organogenic structures occurred on three of four media. Medium I4/D4 was significantly superior to the other two combinations with both genotypes. The occurrence of hyperhydration was the lowest on medium I4/D4.

Example 6:

Effect of cytokinin composition in induction media

The effect of cytokinin composition in the induction media on 2 genotypes using Protocol 2 was studied. Induction media contained 2 mg/l BAP (14), cytokinin omitted (11 1), 1 mg/1 TDZ (112) and 2 mg/1 2ip (113). The differentiation medium was D4. The results are presented in Table 6.

Table 6:

The highest regeneration was obtained on medium 112, the difference was statistically significant compared to 111 and 113 (first genotype), with the second genotype the highest regeneration was obtained on 14 medium. The percentage of hyperhydrated shoots was also low on 112 medium. Omitting cytokinin in the induction media (111) greatly reduced regeneration.

Example 7:

Effect of cytokinin composition of differentiation media

The effect of cytokinin composition in the differentiation media on 4 genotypes using Protocol 2 was studied. Effects of cytokinins were studied at 2 sucrose concentrations. Differentiation media contained 2 mg/1 TDZ (Dl) or 5 mg/1 BAP (D10 and Dl l), induction media were II or 14. The results are presented in Table 7.

Table 7:

Organogenic structures occurred with all studied media and genotypes.

Basal callus structures grew on all media types and with all genotypes. The low sucrose level increased regeneration with all genotypes and lowered the hyperhydration. Example 8:

Effect of light

Formation of organogenic structures in light or in darkness was studied using Protocol 2. Two genotypes and two media (14 and D4) were studied. The results are presented in Table 8.

Table 8:

Organogenic structures occurred with both studied media and genotypes, regeneration in daylight was superior to regeneration in darkness.

Example 9:

Effect of composition of micro and macro elements

Various contents of macro and micro elements in induction and differentiation media were studied in two genotypes using Protocol 2. Induction and differentiation media included BDS medium (I4/D4), B5 medium (I14/D12) or MS medium (I15/D13). The results are presented in Table 9. Table 9:

Organogenic structures occurred on all studied media and with both genotypes. The lowest hyperhydration occurred on media I14/D12.

Example 10:

Determination ofgenotypic effect

Influence of genotype was studied using Protocols 1 and 2 and media Il/Dl . The occurrence of organogenic structures for 12 genotypes of onion are presented in Table 10.

Table 10 (Protocol 1: first embodiment of the claimed process):

Table 11 (Protocol 2: second embodiment of the claimed process):

All tested varieties produced multiple organogenic structures using both embodiments of the claimed process. When flowers were cultured on induction and differentiation media (Protocol 1), a basal callus developed on the bases of some flowers. Occasionally, adventitious shoot regeneration occurred on such calli, and were scored separately from those produced via direct organogenesis. When the ovaries were extracted (Protocol 2) no callus was formed. The induction efficiency differed and differences among cultivars were statistically significant in both embodiments of the claimed process. The highest regeneration rate was 57.9% (Protocol 2) and 45.4% (Protocol 1). It should be noted that the line MSU8155B, exhibiting the lowest regeneration rate using both embodiments, flowered 3 weeks later than the others, so in addition to genotype, an environmental effect (higher temperature in greenhouse) could also have had an impact on the lower regeneration. One half of the varieties responded with similar induction frequencies to both embodiments of the claimed process, while others differed, some responding better using Protocol 1 and some using Protocol 2. The appearance of hyperhydrated shoots also differed among cultivars, and was lower on flowers (Protocol 1) than on ovaries (Protocol 2).

TABLE 1 A

filter sterilised and added to meda a er autocavng

TABLE 11 B differentiation media

Claims

What is claimed is:

1. A process for induction of direct in vitro organogenesis in onion, characterized in that it comprises the steps of:

(i) inoculation of flower buds at a mature stage but before dehiscence on induction media, which contain adequate concentrations of phytohormones, growth regulators and gelling agents for initiation of direct somatic organogenesis, the growth regulators at this stage including an effective mixture of auxins and cytokinins,, and the media containing macro and micro elements, vitamins, inositol, proline, carbohydrates and gelling agents;

(ii) after an appropriate induction time transferring the explants from induction to differentiation media which contain adequate concentrations of cytokinin, macro and micro elements, vitamins, inositol, proline, sucrose and gelling agents till direct organogenesis occurs;

(iii) optional removal of perianth before transfer from induction to differentiation media and culture of extracted ovaries, followed by treatment described in (i) and (ii) leading to induction of direct somatic organogenesis;

(iv) separation of globular embryogenic structures developed as described in (ii) and (iii) followed by transfer of elongated shoots to media for root development or maintenance of compact organogenic structures;

(v) acclimatization of previously (iv) obtained rooted shoots.

2. Process according to claim 1 wherein the said induction media contain media solidifiers gellan-gum, agar or a mixture of agar and gellan-gum.

3. A process according to claim 1 wherein the said differentiation media contain media solidifiers gellan-gum, agar, or a mixture of agar and gellan-gum.

4. A process according to claim 1 wherein the growth time on induction media is from 3 to 12 days.

5. A process according to claim 1 wherein the induction and differentiation media contain sucrose, glucose or maltose as a source of carbohydrates.

6. A process according to claim 1 wherein the said induction and/or differentiation media contain 25-100g/l of sucrose.

7. A process according to claim 1 wherein the said induction media contain cytokinin and 2,4-dichlorophenoxyacetic acid or picloram. as a sources of auxins.

8. A process according to claim 1 wherein the said induction media contain auxin or auxin and 6-benzylaminopurine, thidiazuron or isopentenyladenin (2ip) as sources of cytokinins.

9. A process according to claim 1 wherein the said differentiation media contain thidiazuron or 6-benzylaminopurine as sources of cytokinins.

10. A process as claimed in claim 1 wherein the said induction is being established in light or in darkness.

1 1. A process as claimed in claim 1 wherein the said differentiation is being established in light or in darkness.

12. A process as claimed in claim 1 wherein the said composition of macro and micro elements in induction media corresponds to BDS media, B5 media or MS media.

13. A process as claimed in claim 1 wherein the said composition of macro and micro elements in differentiation media corresponds to BDS media, B5 media or MS media.