An in vivo/vitro embryo culture technique

Transcription

1 Hereditas 77: (1974) An in vivo/vitro embryo culture technique ANTHON KRUSE Department of Crop Husbandry and Plant Breeding, Research Station Hojbakkegdrd, Taastrup, Denmark KRUSE, A An in vivo/vitro embryo culture technique. - Hereditas 77: Lund, Sweden. ISSN Received March 8, 1974 Work has been carried out for a number of years on the development of a new and improved embryo culture technique to supplement or replace the traditional in vitro methods. This new method is based on the implantation of hybrid embryos onto naked Hordeum endosperm placed on a normal growth medium. In 1972 this method was used in the hybridization of HordeumX Triticum and 28 (70 %) hybrid plants were obtained from 40 embryos. In (50 %) hybrid plants developed from 167 embryos derived from Hordeum X Agropyrum crosses. The survival rate in Hordeum X Secale crosses has been increased to 3 M O % by the use of this method compared to about 1 % with traditional in vitro embryo culture. An experiment with barley embryos showed that all embryos mm survived in vivo/vitro culture independent of the composition of the growth medium, whereas similar embryos subjected to in vitro culture showed normal development only when grown on a complete medium supplemented with 25 % coconut milk. It is concluded that the composition of the basic medium is still of importance in in vivo culture of hybrid embryos on Hordeum endosperm. Anthon Kruse, Department of Crop Husbandry and Plant Breeding, Research Station Hajbakkegdrd, DK-2630 Taastrup, Denmark Previous embryo culture methods have been based on the in vitro growth of embryos on a medium comprising inorganic salts and organic compounds in suitable mixtures aimed at promoting normal growth and development in otherwise inviable embryos. The present author has previously employed a traditional in vitro culture technique for growing hybrids, but with limited success; only two of 169 cultured HordeumX Secale hybrid embryos, together with one monoploid barley plant, developed into mature plants (see KRUSE 1967). This work with interspecific and intergeneric hybridization has since been continued and intensified, and attempts have therefore been made to test other media and to devise new methods. In work of this type the achievement of new hybrid combinations is of little value if the resultant hybrid embryos cannot be induced to survive and develop into mature hybrid plants. No attempt will be made in this report to evaluate or select amongst the numerous published in vitro culture media. One point should, however, be emphasized; namely that all of the known media are supposed to simulate the natural conditions under which the often extremely weak hybrid embryos would have developed, had there not existed massive sterility barriers to hybrid formation. This approach led the author to the report of ZIEBUR and BRINK (1951) on the successful use of barley endosperm and endosperm extract for growing young ( mm) barley embryos in vitro. ZIEBUR and BRINK also attempted to grow Raphanus sativus and Capsella bursapastoris embryos on the same medium and obtained equally positive results. In these experiments entire endosperms were placed aseptically on the surface of the culture medium without direct contact with the experimental embryos. In parallel experiment the endosperm was macerated, extracted with water and dialysed. The resultant extract was then admixed with the culture me- dium. ZIEBUR and BRINK (1951) write (p. 254) that: the activity of barley endosperm in stimulating growth of barley embryos in vitro may be attributed to relatively small molecules, since the active principle can pass through a cellofan dialyzing membrane. The present author has repeatedly employed Hordeum vulgare as maternal partner and Secale, Triticum, Agropyrum and other genera as pollen sources, and thus attempts were made to culture

2 220 ANTHON KRUSE hybrid embryos obtained from these crosses directly on immature maternal endosperm. Endosperm generally contains a growth medium which, while specific for each plant species, cannot be replaced or surpassed by artificial in vitro growth media. In this connection POPE (1944) has shown that by means of his vivipary technique quite young (1 week old) Hordeum embryos can be induced to germinate in the ear and develop into normal plants only 15 days after pollination. Thus an immature endosperm can provide sufficient nutrients and induce immediate embryo germination. The metabolic cycle in germinating barley has been investigated and described by JONES and ARMSTRONG (1971) who showed that the initiation of germination is caused by gibberellic-like substances which are secreted by the coleoptile and scutellum and which activate the aleuron protein to produce alpha-amylase. This important enzyme hydrolyses the endosperm starch to maltose and glucose which constitute part of the nutrient and energy source for the germinating embryo, Materials and methods The omission of an in vitro phase by directly transplanting hybrid embryos onto a normal endosperm in active growth by substituting the normal embryo with a hybrid might well be possible. Such a procedure, however, would demand a high degree of accuracy and, due to the diminutive size of the hybrid embryos, would have to be carried out under a microscope and under sterile conditions. The hybrid embryo can also be transplanted to an endosperm which has been transferred to a normal growth medium. An immature endosperm dissected out of the maternal partner and traqsferred to a growth medium is capable of continuing growth, but an embryo germinating on this endosperm will initiate the endosperm metabolic cycle causing the cessation of endosperm growth and its partial exhaustion during the germination period. 1. Basic in vitro media The author's incomplete medium (AK) KCI 65 mg/l KNO, NH4NO3 Ca(NO3) B NaH,PO, KHiPO4 MgS04 50 mg/l 50 mg/l 70 mg/l Adenine I-inositol 40 mg/l Kinetin 0.5 mg/l IAA (indole-acetic-acide) 5 mg/l TOMASZEWSKI and KUBOK (1968) (T+ K) Macro: (NH,)aS g/l (0.25)' KNO, 0.25 g/l Ca(HBP04), 0.20 g/l (0.22 KH,PO,)' Ca(N03), 0.15 g/l MgSO g/1 KCl 0.20 g/l (0.25)' Micro: MnCI, 0.15 mg/l H3B mg/l cuso, 0.05 mg/l ZnSO, 0.30 mg/l (NHJa MOO, 0.15 mg/l Vitamins: B, Thiamin 10 mg/l B8 Riboflavin 7 mg/l B, Pyridoxin 5 mg/l H Biotin PP Nicotinic 5 mg/l acid C Ascorbic 5 mg/l acid 50 mg/l Pantothenic acid 7 mg/l 1 Changed in 1972 after personal communication with Dr. Tomaszewski The above media were employed in the following proportions: AK 1: 10 T+ K macro 1: 10 T+ K micro 1: 100 T+ K vitamins 1: 10 The final composition of the basic medium was thus: 50 ml AK basic solution per I 50 ml T+ K macro solution per 1 10 ml T+ K micro solution per I 70 ml T+ K vitamin solution per 1 Herediias 77, 1974

3 EMBRYO CULTURE TECHNIQUE 221 To this basic medium was added 8 g Bacto-agar (Difco), 30 g saccharose and 3 ml 1 % ferric citrate per litre. Coconut milk has been employed for a number of years, % admixed with the basic medium. The sugar content of the coconut milk was approx. 5 % and ph The final basic medium is thus a modified combination of the media devised by Tomaszewski and the author respectively. Macro compounds and certain biological reagents not included in the Tomaszewski mixture have been taken from the author s medium, whereas Tomaszewski s micro and vitamin mixtures were more comprehensive than those of the author. 2. In vivo endosperm The N content of the various components of barley grains (var. Sultan) were examined with the following results: N content of mature grain 1.95 % N content of 8 ears with 5 grains, totalling 40 grains approx. 14 days old, without hull or embryo 1.59 % N content of 8 earsx 10 grains from each ear, naked endosperm, without pericarp or embryo 1.32 % N content of pericarp of above 8X 10 grains 2.07 % Endosperm ph= These results show that a 14 days old grain has a lower N content than a mature grain, and that compared with the pericarp (aleuron layer) the N content of naked endosperm is considerable, due probably to the fact that nitrogen deposition in the aleuron layer is far from complete after 14 days. The endosperm employed as growth medium must not be too young. The best results have been obtained with endosperm 5-2 days older than the 9-12 days old embryos. This is also in agreement with ZIEBUR and BRINK S (1951) conclusion that older endosperm is most efficient. 3. Procedure For embryo culture 20 ml glass vials with screw lids are employed, each containing approx. 12 ml culture medium. The basic medium is boiled in a 0.5 or flask for min, coconut milk added if required, and the solution made up to 500 or lo00 ml and ph adjusted to approx The medium is then poured into the required number of vials, the lids loosely screwed on, and sterilization accomplished by autoclaving at 2 atm for approx. 20 min. Filter papers (diameter 8 cm) for use in transferring endosperm and embryos to the medium are placed in a suitable number of airtight paper bags (e.g., 10 bags each with 3-5 filter papers), sealed with clips, and sterilised together with clean glass dishes placed in similar paper bags and 100 ml flasks containing distilled water, the necks of which are closed by inverted, 50 ml glass beakers. After sterilisation the lids are screwed tightly onto the culture vials which are subsequently placed at an angle of 45 in order to provide an ellipsoidal surface for optimum illumination. The working area must be clean and draughtfree. Although a sterile room is to be preferred, the present work was carried out in a normal laboratory and only few infected cultures were obtained. A solution of 0.1 % HgCls is employed for disinfecting seeds, while benchtop, microscope, equipment and hands are cleaned by wiping with ethanol. The transplantation is commenced by disinfecting the dehulled seeds, the endosperm donors, for 5-7 min. The culture vials are then prepared by wiping with ethanol, the lid removed, and the vial inverted on the clean benchtop. In addition to a microscope, a gas flame, scissors, needle, scalpel (Gillette E-shape) and forceps are employed. The top of a paper bag containing a sterilised glass dish is then cut off in such a manner as to leave a quadrant-shaped bag around one corner in which the glass dish can remain protected from contamination during use. The dish is filled with sterile HpO into which the HgCI2- treated seeds are immersed. A sterile filter paper moistened with sterile water is then placed on the microscope stage. The seeds are taken singly and using a flamesterilised scalpel and needle the embryo is dissected out under a magnification of 1OX. Using the side of the scalpel blade the sterile endosperm can then be pressed out of the pericarp with the germ end leading (Fig. 1). The endosperm is placed on the medium surface either singly or 2-4 endosperms per vial. The vials must be held at an angle with the aperture downwards and replaced on the bench in the same position. The endosperm should preferably be intact, with the former sur-

4 222 ANTHON KRUSE C in a growth cabinet with a light intensity of about 3000 lux for 16 hours and a dark period of 8 hours. During germination embryos which have been inaccurately placed on the endosperm can be adjusted with a sterile needle, and if necessary embryos from older cultures (approx. 4 weeks) can be transferred to new cultures. After successful germination (Fig. 2) hybrid plants with one to several roots and 2-3 leaves can be transplanted after 3-4 weeks to a sterile soil consisting of /2 sand : /2 sphagnum and liquid inorganic fertiliser applied. The plants are protected from direct sunlight by means of plastic sheeting for the first few days after planting. Fig. 1. Dissection of endosperm. Right: normal IS days old seed. Centre: dehulled seed, embryo removed. Left: pericarp and extruded endosperm ready for use. Scale in mm. face of embryo attachment undamaged. When a sufficient number of endosperms have been transferred to culture vials the transfer of hybrid embryos can be commenced. Although the author has transplanted hybrid embryos only a few days old, the best results are obtained with embryos 9-12 days old. The small and underdeveloped hybrid seeds are dehulled, treated with 0.1 % HgCI, for 1-2 min and placed in a glass dish with sterile water. The embryos are then rapidly dissected out at a magnification of 16X and placed in the correct position at the site of the natural embryo (Fig. 2). The vial is then closed with a flame-sterilised porous cellulose stopper ( steri-prop ). It is important that no trace of ethanol, etc., remains on the needle or scalpel used for dissection, and that both are allowed to cool to room temperature after flame-sterilisation. Microscope light sources often emit considerable heat and the lamp must be arranged at such a distance from the embryos as to avoid damage. If the hybrid embryos are extremely small ( mm), it can be of advantage to place up to 5-10 on a single endosperm. The author has found that whereas the transplantation of one very small embryo often fails to start germination, several embryos are able to initiate the endosperm metabolic cycle. The embryo cultures are maintained at Culture experiments with barley embryos Although an experiment with barley embryos grown in vivo and in vitro cannot give a complete answer as to whether an embryo culture based on natural endosperm is superior to normal in vitro methods, the author has grown 1.O- 1.2 mm and 50.2 mm barley embryos (var. Emir) both on endosperm and in vitro on agar in the same cultures. Each culture vial thus contained four treatments, and each experiment represented a new replication. In each replication the endosperms were derived from the same ear, and similarly the embryos used within groups were obtained from the same ear and were thus of the same size and age. The experimental design is indicated in Table 1. The larger (a) embryos showed good germination on all media, both when implanted on endosperm and when placed on agar. None of the small (b) embryos germinated on agar in treatment U, but all showed slow germination when implanted. In treatment OK 1 out of 3 small (b) embryos germinated on agar, whereas all germinated (1 slowly) on endosperm. In treatment 25K with 25 % coconut milk both large and small embryos germinated normally, independent of whether placed on agar or on endosperm. While there can be no doubt as to the positive effect of endosperm as a growth medium especially for small embryos, media comprising only saccharose and agar were found to be inadequate, also when embryos were implanted on endosperm. The complete medium was reasonably satisfactory both with and without endosperm, but the best results were obtained when the medium was supplemented with 25 % coconut milk.

5 EMBRYO CULTURE TECHNIQUE 223 Fig. 2 a-f. Embryo culture. - a-c: Hordeum vulgarex Triticum aestivum; a: hybrid seeds 12 days old, a normal seed to the right; b: hybrid embryos implanted on endosperm, size p; c: same embryos photographed one week later. - d-f: Hordeum vulgarex Elymusarenarius; d: hybrid seeds 12 days old, a normal seed to the left; e: hybrid embryos implanted on endosperm, size p; f: same embryos photographed one week later. Scale in mm. Hereditas 77, I974

6 224 ANTHON KRUSE Table I. In vivo/vitro embryo culture experiments Media: U1 OK2 25K3 Embryos: a4 - b5 a4 - b5 a4 - b5 1. exp. Endosp. + (+) (+) Agar (+) 'I..I. 2. exp. Endosp. (+) Agar + 'I. 3. exp. Endosp. (+) (+) + Agar + 'I. +.I U = medium comprising only 0.8 % agar + 3 "/o saccharose 2 OK =complete medium as described, without coconut milk 3 25K= complete medium as described, with coconut milk 4 a = 1.G1.2 rnm embryos 5 b Z0.2 mm embryos + = normal germination (+) = slow germination 'I. =no germination Results and discussion Although there has been no new development in the field of embryo culture since this technique was first devised, the present author has during recent years utilized the natural growth medium, the endosperm, in the culture of underdeveloped hybrid embryos, an approach which ZIEBUR and BRINK (1951) followed more than 20 years ago. The in vivo/vitro embryo culture method described here was used in a HordeumX Triticum programme (KRUSE 1973) and resulted in 28 hybrid plants from a total of 40 cultured embryos. In recent work with Hordeum X Agropyrum hy- brids (KRUSE 1974, in prep.) 83 hybrid plants have been obtained from 167 cultured embryos. In earlier experiments employing in vitro embryo culture, only 2 HordeumX Secale hybrids developed from 169 hybrid embryos. Using the in vivo/vitro technique per cent of these embryos can now be induced to germinate and develop into hybrid plants. These results may be due in part to the GA treatment employed by the author in the production of hybrid embryos (cf. the influence of GA on seed germination in Hordeum as demonstrated by JONES and ARM- STRONG in 1971). In the experiment described above with in vivo/ vitro culture of large and extremely small Hordeum embryos it was found that all of the small embryos ( 50.2 mm) were able to develop on endosperm, whereas only 4 out of 9 similar embryos survived after in vitro culture, depending on the composition of the medium. Acknowledgments. - The author wishes to thank Bent Toft Viuf for performing the N analyses of different grain components and Brian A. Dennis for translating this report. Literature cited JONES, R. L. and ARMSTRONG, J. E Evidence for osmotic regulation of hydrolytic enzyme production in germinating barley seeds. -Plant Physiol. 48: KRUSE, A Intergeneric hybrids between Hordeum vulgare and Secale cereale. - K. Vet. og Landbohojsk. Rrsskr.. p Hordeum X Triticum hybrids. - Hereditas 73: POPE, M. N Some notes on technique in barley breeding. - J. Hered. 35: TOMASZEWSKI, Z. and I. KUBOK Hodowla thanek merystematycmych kielkow ziemniaka jako metoda otrzymania bezwirusowych roslin. - Hodowla roslin, aklimatyzacja i nasiennictwo 12 (4): ZIEBUR, N. K. and BRINK, R. A The stimulative effect of Hordeum endosperms on the growth of immature plant embryos in vitro. -Am. J. Eot. 38: Publication no. 174 from Research Station Hojbakkeghrd, Taastrup, Denmark