CA1292960C - Process for increasing free pool lysine content in maize - Google Patents
Process for increasing free pool lysine content in maizeInfo
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- CA1292960C CA1292960C CA000553057A CA553057A CA1292960C CA 1292960 C CA1292960 C CA 1292960C CA 000553057 A CA000553057 A CA 000553057A CA 553057 A CA553057 A CA 553057A CA 1292960 C CA1292960 C CA 1292960C
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Abstract
TITLE OF THE INVENTION
PROCESS FOR INCREASING FREE POOL
LYSINE CONTENT IN MAIZE
ABSTRACT OF THE DISCLOSURE
The present invention is directed to a process for increasing the free pool lysine content of individual maize seeds and for increasing the average free pool lysine content of seeds produced by a maize plant. The process, in general, comprises initiating callus from maize tissue, selecting for callus tissue resistant to S-2-aminoethyl-L-cysteine, and regenerating plants from the selected callus. In addition, the callus may be maintained before or during the selection scheme, or in place of the selection scheme.
PROCESS FOR INCREASING FREE POOL
LYSINE CONTENT IN MAIZE
ABSTRACT OF THE DISCLOSURE
The present invention is directed to a process for increasing the free pool lysine content of individual maize seeds and for increasing the average free pool lysine content of seeds produced by a maize plant. The process, in general, comprises initiating callus from maize tissue, selecting for callus tissue resistant to S-2-aminoethyl-L-cysteine, and regenerating plants from the selected callus. In addition, the callus may be maintained before or during the selection scheme, or in place of the selection scheme.
Description
129296;0 TITLE OF THE INVENTION
PROCESS FOR INCREASING FREE POOL
LYSINE C~NTENT IN MAIZE
BACKGROUND OF THE INVENTION
Field of the Invention The present invention relates to a process for increasing the free pool lysine content in maize through selection in tissue culture .
Description of the Prior Art Cereal grains are a major source of vegetable protein. Maize ( Zea mays L . ) contributes approximately one-fourth of the total cereal protein produced. However, maize has a low content of certain essential amino acids, especially Iysine, methionine and typtophan. As a result, maize ln the diet must be supplemented 15 with food containing these amino acids in order to provide a balanced diet, One goal of plant breeding in maize has been to increase the amount of Iy6ine and tryptophan present in the seed.
At least two approaches can be visualized to accomplish an increase in these amino acid6. The first approach is to increase the Iysine 20 or tryptophan content in the proteins found in the maize kernel.
The second is to increase the free pool ~endogenous) Iysine or tryptophan content within the maize kernel.
The first significant breeding results with regard to changes of the protein composition of maize kernels in the direction desired 25 was the discovery by Mertz, E.J., et al. (Science 145, 279 (1964)) that the protein composition of maize endosperm could be dra6tically changed by a single gene (opaque-2). In the following year, the same authors reported a second mutant gene (~loury-2~
which changed the protein composiffon of maize endosperm in a 30 similar way (Nelson, O.E., et al., Science 150, 1469 (1965)). It was found that, besides similar changes in l~sine and tryptophan~
floury-2 also has a higher methionine content.
1~9Z96~
.
It has been demonstrated (Nelson, O.E., Genet.Agron. 21, 209 (1967)) that the different amino acid composition of the two maize mutants i8 chieny due to the modification of the relative amounts of protein fractions , i . e ., a partial suppression of the 5 prolamine and its replacement by other fractions rich in lysine and tryptophan .
Considering the agronomic performance and especially the yield, it seems that opaque-2 is somewhat inferior to normal maize, chiefly due to its lighter kernels. In populations where opaque-2 10 had been introduced into inbred lines, it has been found that the percentage weight loss of opaque-2 kernels as compared to normal sibs varied from less than 596 to 40~6, depending on the inbred line.
Although opaque-2 stocks produce in general smaller and 15 lighter kernels than normal stocks, it has been pointed out that it would be unsafe to conclude that opaque-2 types have necessarily a lower yield than normal i80genic types. The data indic~te that modifier genes affect kernel size in opaque-2 homozygotes;
therefore appropriate selection in segregating populations should 20 be effective in improving that trait. Other researchers are of a similar opinion and point out ~hat the density of opaque-2 kernels is dependent on the genetic background which would mske it possible to select lines in which opaque-2 shows higher densities.
The opaque-2 gene has been incorporated into corn hybrids 25 which commonly had a lower yield than their normal hybrid counterparts. Recently, however, improvements in yield have spurred renewed interest in high lysine corn usage.
Although much effort has been expended to increase the lysine content of maize proteins, very little effort has gone 30 towards the increase of lysine content in maize by increasing the free pool Iysine content.
Plant regeneration from cells in culture i8 essential for the application of somatic hybridization, for the production of new varieties through somoclonal variation or in vitro selection, and for 35 the use of genetic engineering in producing new varieties.
Although plants can be regenerated from tissue culture of several lZ~Z9ti~
varieties of corn, there are many varieties for which this has not been accomplished using similar techniques.
In recent years, plant cell culture successes have had a considerable influence on the understanding of the respective roles 5 of cell and organism in control of plant growth and development.
Isolated plant cells have been shown to be amenable to ~n vitro culture and complete plants have been regenerated from cultures derived from somatic tissues, either directly via somatic embryogenesis or indirectly via organogenesis. Generally, the 10 regeneration pathway of choice is determined empirically by the manipulation of extrinsic factors, especially growth regulators.
Early investigations of certain plant species have ~uggested that exogenous auxin concentration is a major factor controlling somatic embryogenesis, such that its reduction leads to the initiation of 15 embryoid formation, In other species, expo~ure to a definite balance of auxin and cytokinin leads to the occurrence of organogenesi~ (shoots, then roots). Although several genotypes of corn have been regenerated using these techniques, no process i8 generally applicable to most genotypes of corn. Many genotypes 20 remain extremely difficult, if not impossible, to culture using the prior processes.
The process which has become the standard gystem for corn tissue culture i8 described by Green et al ., Crop Science 15 , 417 (1975). In this process, immature embryos were plated onto a 25 calluæ induction medium which comprises the MS mineral salts, Straus vitamins and amino acids ( glycine, asparagine, niacin, thiamine, pyridoxine and pantothenic acid), 2% sucrose, 0.8~6 agar and a hormone ~elected from 2, 4-dichlorophenoxyacetic acid (2, 4-D), p-chlorophenoxyacetic acid (p-CPA), -naphthaleneacetic 30 acid (NAA), 2isopentyladenine (2-ip) or mixtures thereof.
Hormone concentrations which were useful were 2 mg/l 2,4-D and a mixture of 1 mg/l 2, 4-D, 4 mg/l NAA and 0 . 05 mg/l 2-ip .
Plantlets were regenerated by subculturing the callus on medium containing reduced hormone concentrations. Regeneration was then 35 accomplished on medium containing 0.25 mg/l 2,4-D or a mixture of mg/l NAA and 0 . 05 mg/l 2-ip, re~pectively . ~ll culturing wa~
96~
conducted in a 16 hour light/8 hour dark cycle for 3-4 week intervals before transfer. This reference reports that callus induction did not occur in one of five genotypes tested.
Similar results with different n~3dia have been den~nstrated by Fr~eling et al., Maydica 21, 97 (1976); Vasil et al., eor.
Genet. 66, 285 (1983); E;dallo et al., Maydica 26,39 (1981); Lu et al., ~eor. ~p1. Genet. 62, 109 (1982); Gegenbach, Proc. Nat.
Acad. Sci. USA 74, 5113 (1977); and Green et al., Cr ~ Scienoe 14, 54 (1974). Ihe latter reference also den~nstrates genotype effects on ca11us inductian.
Although this procedure hss generally been unsuccessful for regenerating plants from al] maize genotypes, the regeneration of most genotypes is now possible through the substitution of dicamba for 2, 4-D in the media . See published European Application No .
0 177 738 snd Duncan et al.f Planta 165, ~22 (1985).
. _ SUMMARY OF THE IN~7ENTION
. .
The present invention is directed to a process for increasing the free pool lysine content in maize seed and for increasing the average free pool lysine content of seeds produced by a maize plant. The process comprises the regeneration of plants from tissue in culture which has been selected through the use of a Iysine anslog, More specifically, the present process comprises the steps of:
(a) culturing tissue obtained from a maize plant on a callus induction medium comprising mineral salts, vitamins, sucrose and a hormone to form callus;
(b) subculturing said callus on selection medium comprising mineral salts, vitamins, sucrose, S-2-aminoethyl-L-cysteine (AEC) snd a hormone to produce selected callus; snd (c) sub&ulturing said selected callus on regeneration medium comprising mineral salts, vitamins snd ~ucrose to regenerate plants.
Alternatively, the prscess can comprise the additional step of subculturing the callus maintenance medium comprising mineral lZ9Z960 salts, vitamins, sucrose and a hormone before subculturing on the selection medium.
The plants are gro~ n to produce seed (Rl) which can be used to produce further generations of plants.
The present invention is directed to a pro~es~ for increasing the free pool lysine content in maize seed and fo- increasing the average free pool lysine content of seeds produced by a maize plant. The process utilize~ tissue culture selectior of maize tissue 10 with AEC as described further below.
The present invention is directed to maize seeds which have a *ee pool Iysine content of at least about 500 1Jg per gram dry seed weight. Preferably, each seed has at least about 600 ~g of free pool lysine per gram dr~ seed welght. In the most preferred 15 embodiment, each seed has at least 700 ~g of free pool lyEine per gram dry seed weight.
The present invention is further directed tt maize plants, each of which produces seeds having an average free pool lysine content of at least about 325 ~g per gram dr~ seed weight.
- 20 Preferably, each plant produces seeds having an a~erage free pool lysine content of at least about 400 llg per gram d~y seed weight.
It is most preferred that the average free pool lysine content be at least about 500 l~g per gram dry seed weight. It s~.ould be evident that "average free pool lysine content" refers to the average of 25 the free pool lysine content of the individual seeds produced by the maize plant.
The initial regenerated plants (Ro~ are produ~ed from tissue culture generally by regenerating plants from 'dssue in culture which has been selected through the use of a ly~;ine analog, S-2-30 aminoethyl-~-cysteine (AEC). It is also possible to isolate plants which meet the desired characteristics without the use of an AEC
selection scheme. The following generations of plants can be produced by germinating the seed (Rl seed~ produced by the Ro plants. The Rl plants can be self-pollinated to produce R2 seeds.
lZ9Z960 Alternatively, the Rl plants can be incorporated into a breeding program to produce hybrids, inbreds, etc. Any generation of plants having the initial R1 seed as a parent can be used to develop hybrids or inbreds, etc.
For example, if possible, the regenerated plants or their progeny are self-pollinated. In addition, pollen obtained from the regenerated plants or their progeny is crossed to seed grown plants of agronomically important inbred lines. In some cases, pollen from plants of these inbred lines is used to pollinate regenerated plants or their progeny. The trait is geneticall~-characterized by evaluating the segregation of the trait in first-and later-generation progeny. The heritability and expression in plants of traits selected in tissue culture are of particular importance if the traits are to be commercially useful.
The commercial value of maize having a~ increased free pool Iysine content is greatest if many different hybrid combinations are available for sale. The farmer typically grows more than one kind of hybrid based on such differences as maturity, standability or other agronomic traits. Additionally, hybrids adapted to one part of the corn belt are not adapted to another part because of differences in such traits as maturity, disease and insect resistance. Because of this, it is necessary to breed increased free pool Iysine content into a large number of parental lines so that many hybrid combinations can be produced.
Adding the increased free pool Iysine genotype to agronomically elite lines is most efficiently accomplished if the genetic control of free pool lysine content is understood. This requires crossing increased free pool Iysine plants with low free pool ly~ine plants and studying the pattern of inheritance in segregating generations to ascertain whether the trait is expressed as a dominant or recessive, the number of genes involved, and any possible interaction between genes if more than one is required for expression. This geneffc analysis can be part of the initial efforts to convert agronomically elite, yet }ow free pool 3~ lysine content, lines to increflsed free pool lysine lines.
lZ~Z9f~0 A conversion process (backcrossing) is csrried out by crossing the original increased free pool lysine line to normal elite lines and crossing the progeny back to the normal parent. The progeny from this cross will segregate such that some plsnts carry 5 the gene(s) responsible for increased free pool lysine, whereas some do not. Plants carrying such genes will be crossed again to the normsl parent, resulting in progeny which segregate for increased free pool lysine ar.d normal production once more. This is repeated until the original normal parent has been converted to 10 sn increased free pool line, yet possesses all other importsnt attributes as originally found in the normsl parent. A separate backcrossing program is implemented for every elite line that is to be converted to an increased free pool lysine line.
Subsequent to the backcrossing, the new lines and the 15 appropriate combinations of lines which malce good commercial hybrids are evsluated for increased free pool lysine content as well as 8 battery of important agronomic traits, Increased free poo]
lysine lines and hybrids are produced which are true to type of the originsl normal lines and hybrids. This requires evalustion 20 under a range of environmental conditions where the lines or hybrids will generally be grown commercially. For production of increased free pool lysine maize, it may be necessary that both parents of the hybrid seed corn be bomozygous for the increased free pool lysine characterO Parental lines of hybrids that perform 25 satisfactorily are increased and used for hybrid producffon using standard hybrid seed corn production practices.
In general, the process to produce Ro plants comprises (a) culturing maize tissue on a medium to produce callus; (b) selecting callus tissue which is resistant to AEC in the culture medium, and 30 (c) regenerating plants from the ~elected callus. The callus can optionally be maintained on a maintenance medium prior to tran~fer to a selecffon medium. The regenerated plants are matured and self-pollinated to produce Rl seeds. R2 seeds are produced by self-pollinating Rl plants germinated from ~1 seeds. Additional 35 generations can be obtained in like manner.
lZ9Z9~iO
Although the process to increase free pool lysine content generally uses a selection scheme with AEC, it is possible to produce maize seeds and plants having the desired characteristics without selection. In this instance, the callus is maintained on a 5 maintenance medium rather than cultured on a selection medium.
This process is not as efficient , i . e ., it has a lower frequency of success than selection with AEC.
Within the general process outlined above, several tissue culture sequences can be utilized to produce maize seeds and 10 plants having the desired characteristics. The preferred sequences can be summarized as follows:
In the first preferred sequence, callus is induced on a callus induction mediu~ as described below. The callus is then transferred to maintenance medium for 6-9 passages before transfer 15 to regeneration medium. The plants are then transferred to soil.
No selection is performed àt any step during this sequence.
In a second preferred sequence, callus is induced on a callus induction medium as described below. The callus induction medium could contain 0-0.2 mM AEC. If no AEC is contained in the callus 20 induction medium, the callus is transferred to maintenance medium for 2-6 passages before it is transferred to selection medium. If the callus induction medium contains AEC, the callus is then transferred to selection medium rather than to maintenance medium.
The callus is transferred to selection medium for 3-8 passages 25 before transfer to regeneration medium. The concentration of AEC
i8 increased step~ise from 0 .1 to 3 . O mM, preferably from 0 .1 to 1.0 mM or from O.S to 2.0 mM, through the passages. In~an alter-native, the AEC concentration is dropped to zero after a stepwise increase in concentration, i.e., transfer to a maintenance medium, 30 and then raised to 1.5-2.5 mM, preferably 1.5-2.0 mM, in the next transfer before proceeding to regeneration medium. 2-4 passages, preferably 3 pas~ages, on regeneration medium may be performed prior to transferring the plants to the soil. The regeneration medium may contain 0-0.5 mM AEC. It is preferred to utilize AEC
35 in the regeneration medium and to lower its concentration stepwise through the passages on the regeneration medium.
lZ929~0 g The third preferred sequence is silailar to the second sequence except that the concentration of AEC is increased stepwise from 1. 0 to 3 . 0 mM, preferably from 1. 0 to 2 . 0 mM, during the passages on the selection medium.
In a fourth preferred sequence, callus is induced on a callus induction medium as described above. The callus induction medium could contain 0-0.2 mM AEC. If no AEC is contained in the callus induction medium, the callus is transferred to maintenance medium for 2-6 passages before it is transferred to selection medium. If the callus induction medium contains AEC, the callus is then transferred to selection medium. The callus i8 transferred to selection medium for 3-8 passages before transfer to regeneration medium . The concentration of AEC may be 0 . 25-0 . 5 mM for 1-3 passages, preferably 2 passages, and then it is raised to 2.0-3.0 mM, preferably 2 . 0 mM, for 1-2 passsges, preferably 1 passage.
The AEC concentration is then lowered to û.5 mM and increased stepwise to 1. 0-1. 5 mM over 3-6 passages, preferably 4 passages .
In an alternative, the AEC concentrat~on is dropped to zero after a stepwl8e increase in concentration, i.e., transfer to a maintenance medium, and then rai~ed to 1.5-2.0 mM in the next transfer before proceeding to regeneration medium. 2-4 passages, preferably 3 passages, on regeneration medium msy be performed prior to transferring the plants to the soil. The regeneration medium may contain 0-0 . 5 mM AEC . It is preferred to utilize AEC in the regeneration medium and to lower its concentration stepwise through the passages on the regeneration medium.
The preferred sequences described above can be modified in accordance with the teachings herein and within the skill of the art. Several modifications include (a) reduced number of transfers on certain of the media, (b) addition of other media to the -sequence, (c) use of other basal media well known in the art, (d) the deletion of the transfers to maintenance media, (e) addition of more traTufers to maintenance media du~ng the overall selection scheme, (f) reduced passage duration, (g) increased number of transfers on each medium, and (h) modification of AEC in sequence.
lZ929~0 The plant tissue which is preferred for use in iniffation of callus is the immature embryo. It is preferred to use an immature embryo from a plant w hich has an elevated level of lysine. Such maize lines include lines 1007, 1008, 1010 and 1012 of Crow's 5 Hybrid Corn Company, Milford, Illinois. The imm~ture embryos are isolated from the cob at approximately 10-17 da~s post-pollination when the embryos are 1.0-2.0 mm, preferabl~ mm, in length.
The cob is harvested and surface-sterilized. ~he immature embryos are isolated from each kernel. The embryos are plated onto callus 10 induction medium which may or may not contair; AEC. The embryos are plated so that the embryo axis is in contact with the medium, i.e., the scutellar side is up.
The callus induction medium comprises mineral salts, vitamins and sucrose. The mineral salts comprise macroelements and 15 microelements. The macroelements used in the callus induction medium may be the following compounds: magnesium sulfate, calcium chloride, monopotassium phosphate, potassium nitrate and ammoniùm sulfate. The microelements contained in the callus induction medium are: boric acid, manganese sul'ate, zinc sulfate, 20 potassiurn iodide, iron (Il) sulfate, disodiu=-ethylenediamine-tetracetic acid (EI)TA), sodium molybdate (VI), copper (II) sulfate and cobalt chloride. This combination of mineral calts is known in the art ~ the N6 mineral salts, which have been modified to contain mineral salts of copper, cobalt and mol~bdenum. Other 25 combinations of mineral salts may also be used as long as they do not adversely affect callus induction. Examples of combinations of mineral salts include, but are not limited to, ~S, Heller, Nitsch and Nitsch~ B5 and White.
The preferred amounts of the macroelements and microelements 30 used to prepare one liter of the callus induc~on medium are as follows: 185 mg magnesium sulfate heptahydrate, 166 mg calcium chloride dihydrate, 400 mg monopotassium phosphate, 2830 mg potassium nitrate, 463 mg ammonium sulfate, 1.6 llig boric acid, 4.4 mg manganese sulfate monohydrate, 1.5 mg rinc sulfate hepta-35 hydrate, 0.83 mg potassium iodide, 27.8 mg iron (II) sultateheptahydrate, 37.3 mg disodium-EDTA, 0.25 mg ~odium molybdate lZ9Z960 (VI) dihydrate, 0.025 mg copper (Il) ~ulfate pentahydrste and 0.0a5 mg cobalt chlo~qde hexahydrate.
The callus induction medium ~so contains vit~;nins. ~he vitamins used include myo-inositol, nicotinic acid, glycine, X pyridoxine, thiamine, and pantothenate. The amounts of ~ itamins used to prepare one liter of the callus induction medium are ~s follows: l00 mg myo-inositol, 0.5 mg nicotinic acid, 2 mg glycine, 0.5 mg pyrido~ne hydrochloride and ~.0 mg thiamir~e hydrochloride and 0.25 mg calcium pantothenate.
The first medium contains 2-6%, preferably 3%, ~ucrose and a gelling agent such as agar or GelriteTM (tr~demark, Kelco Commercial Development, P.O. Box 23076, San Diego, California).
It is preferred to use Bacto-Agar st a concentration of about 0.78%. The medium has a pH of 5.5-6.0, w;th a preferred pH of 5.8, before autocls~ring.
In sddition to the above components, the callus induction medium also contains a hormone. As used herein, "hormoner is lntended to mean any natural or synthetic compound which has a regulatory effect on plants or plant tissue. Plant hormones include auxins and cytokinins. The hormone which i~ useful for callus inducffon h the present in~.rention is 2,4-D. 2,4-D can be utilized alone or in combination with another hormone such as 2,4,5-trichlorophenoxyacetic scld (2,~,5-T) or zeatin. Other hormones, such ~ those described in publi~hed European application No, 0 177 738 and copending U.S. Patent No.
4, 830, 966, can be usea. The amount of honnone is ~ufficient to insure callus formation. Generally, about 2-3 mg/1 of 2, 4-D is sufficient, with 2 mg/1 preferred.
If zeatin or 2, 4, 5-T are also present, they can be utilized ln an amount of 2-3 mg/1, preferably about 2 mg/1.
The callu~ induction medium may also contain a low concentration of ~EC. It i8 preferred that no ~EC be pre~ent in this medium. If AEC i6 pre~ent, it i~ preferred that a concentratlon of 0.05-0.2 mM be utilized.
The immature embryos are plated on the callus induction medium and cultured in dif u~ed light wlth a photoperiod of 16 lZ'329~0 hours per day for 2-5 weeks, preferably 3-4 weeks. Dulqng this time, the embryo undergoes de-differentiation and callus formation.
After culturing the immature embryo on the callus induction medium, the callus is transferred and subcultured on either a maintenance medium or a selection medium. Generally, the only difference between the maintenance medium and the selection medium is that the latter contains AEC. The callus may be cultured on the maintenance medium for- 3-6 transfers before it is transferred to selection medium. The callus may be cultured on the selection medium for 3-8 transfers before it is transferred to regeneration medium. The callus is transferred to fresh maintenance or selection medium every 10-70 days.
The maintenance and selection media comprise mineral salts, vitamins, sucrose and a hormone in an amount sufficient to maintain the callus. The mineral salts and vitamins are as described for the callus induction medium. As in the callus induction medium, various combinations of mineral salt~ which do not adversely affect the functioning of the medium may be utilized.
The sucrose concentration is 3-6~, preferably 396. The hormone is generally 2,4-D at a concentration of 2-3 mg/l, preferably 2 mg/l, although others can be used as discussed above. Bacto-Agar at 0.78% is used to solidify the medium.
The maintenance and selection media may further contain L-glutamine and citric acid. If these components are utilized, it is preferred to use a concentration of 10 mM L-glutamine and 5 mM
citric acid.
The selection medium further contains AEC. The concentration of AEC in the selection medium may range from 0 .1-3 . 0 mM, preferaMy 0.1-2.0 mM. The concentration of AEC is varied during the selecffon process, as will be described further below.
After completing the selection sequence, the selected callus is placed on regeneration medium. One or more regeneraffon media may be utilized, with transfers to fresh regeneration medium occurring at about 5-40 days. Generally, 2-4 transfers may be Ufflized~
l;~9Z960 The regeneration medium comprises mineral salts, vitamins and sucrose. The mineral salts may be the same as for the callus induction medium, i.e., N6 salts, or they may be the MS mineral salts. It i~ preferred to utilize N6 salts.
The regenerstion medium also contains vitamins. The vitamins which may be utilized are either (a) thiamine, nicotinic acid, pyridoxine and glycine, or (b) thiamine, glycine and myo-inositol.
The (a) vitamins are referred to herein as N6 vitamins plus glycine, and the (b) vitamins are referred to herein as vitamin G.
The amount of vitamins used to prepare one liter of regeneration medium is as follows: (a) for N6 vitamins: 1 mg thiamine hydro-chloride, 0.5 mg nicotinic acid, 0.5 mg pyridoxine hydrochloride and 2 mg glycine; and (b) for vitamin G: 1 mg thiamine hydro-chloride, 2 mg glycine and 100 mg myo-inositol.
It is preferred that the regeneration medium contains 2-6%
sucrose, and the gelling substance may be Bacto-Agar or Gelrite at about 0.78% or about 0.2296, respectively.
The regeneration medium may contain AEC at a concentration of 0 .1-0 . 50 ml~ . It is preferred to reduce the AEC concentration during the transfers which are performed at about 5-40 days during the regeneration cycle. The regeneration medium may also contain a hormone. It i~ preferred to utilize one or more cytokinins if a hormone is used. The hormone may be selected from a mixture of (a) 0.2-0.5 mg/l indoleacetic acid (IAA) and 0 . 4-1 . 5 mg/l benzyl amino purine (BAP), preferably either 0 . 3 mg/l and 1.0 mg/l, respectively, or 0.3 mgll and 0.5 mg/l, respectively, or 0.25 mgll and 1.0 mgll, respectively; (b) 0.2-0.5 mgll IAA and 0.8-1.5 mg/l BAP, preferably 0.3 mg/l and 1.0 mgll, respectively; and (c) 0.1-0.3 mgll 2,4-D, 0.05-0.2 mg/l BAP
and 0.2-0.5 mgll gibberellic acid (GA3), preferably 0.2 mg/l, 0.1 mg/l and 0.35 mg/l, respectively.
After plants have been regenerated and established, they are transferred to cubes which contain one part of potting soil and one part of vermiculite. The gelling substance i8 washed off the plants prior to transfer to the cubes. The plantg are transferred to 12" pots containing potting 80il after about 4-40 days and 12929f~
. .
placed in the greenhouse. All of the culturing described above and that described below in the examples i8 conducted at about 24C with a 16 hour diffused light/8 hour dark cycle.
In several instances, the Rl seeds produced by plants obtained in accordance with the above process showed a decreased free pool lysine content. However, plants germinQted from these Rl seeds produced R2 seeds having an average free pool Iysine content of ~t least 325 lJg per gram dry ~eed weight. Thus, a lo~
free pool lysine content in Rl seed does not indicate that the fin~l 10 desired characteristics cannot be obtained. It demonstrates that ~everal generations of plants may be re~uired to obtain plants and seeds having the desired characteristics.
The present invention will be further described by reference to the following non-limiting examples.
Preparation o~ Solutions The following Atock solutions or solutions were prepared for use in making the media described in further detail below.
1. OPA Reaction Mi~c OPA reaction mlx was prepared b~ dissolving 2.6 g of boric acid in ~0 ml of deionized water. The pH was adjusted to 10.4 with a 45~ golution of KOH~ 200 1ll of 2-mercaptoethanol was added, followed by 300 pl of Brij-35. Brij-3~ ~as added to gtabilize the OPA-lysine complex (Anal.Biochem. 101, 61 (1980)). 80 mg of 25 Fluoropa *(OPh from Pierce) was dissol~ed in I ml methanol and then added to the borate solution. The volume was sdjusted to 100 ml with deionized water. The OPA resction mix was kept at 4C and was di~carded after two weelcs to insure low background peaks.
* Trademark 2. AEC Stock Solution A 500 mM stock solution of AEC was prepared by dissolving 5 . 02 g of AEC in 500 ml of deionized water or by dissolving 1.004 g of AEC in 100 ml of deionized water. The pH was adjusted to 5.8 using lN KOH.
PROCESS FOR INCREASING FREE POOL
LYSINE C~NTENT IN MAIZE
BACKGROUND OF THE INVENTION
Field of the Invention The present invention relates to a process for increasing the free pool lysine content in maize through selection in tissue culture .
Description of the Prior Art Cereal grains are a major source of vegetable protein. Maize ( Zea mays L . ) contributes approximately one-fourth of the total cereal protein produced. However, maize has a low content of certain essential amino acids, especially Iysine, methionine and typtophan. As a result, maize ln the diet must be supplemented 15 with food containing these amino acids in order to provide a balanced diet, One goal of plant breeding in maize has been to increase the amount of Iy6ine and tryptophan present in the seed.
At least two approaches can be visualized to accomplish an increase in these amino acid6. The first approach is to increase the Iysine 20 or tryptophan content in the proteins found in the maize kernel.
The second is to increase the free pool ~endogenous) Iysine or tryptophan content within the maize kernel.
The first significant breeding results with regard to changes of the protein composition of maize kernels in the direction desired 25 was the discovery by Mertz, E.J., et al. (Science 145, 279 (1964)) that the protein composition of maize endosperm could be dra6tically changed by a single gene (opaque-2). In the following year, the same authors reported a second mutant gene (~loury-2~
which changed the protein composiffon of maize endosperm in a 30 similar way (Nelson, O.E., et al., Science 150, 1469 (1965)). It was found that, besides similar changes in l~sine and tryptophan~
floury-2 also has a higher methionine content.
1~9Z96~
.
It has been demonstrated (Nelson, O.E., Genet.Agron. 21, 209 (1967)) that the different amino acid composition of the two maize mutants i8 chieny due to the modification of the relative amounts of protein fractions , i . e ., a partial suppression of the 5 prolamine and its replacement by other fractions rich in lysine and tryptophan .
Considering the agronomic performance and especially the yield, it seems that opaque-2 is somewhat inferior to normal maize, chiefly due to its lighter kernels. In populations where opaque-2 10 had been introduced into inbred lines, it has been found that the percentage weight loss of opaque-2 kernels as compared to normal sibs varied from less than 596 to 40~6, depending on the inbred line.
Although opaque-2 stocks produce in general smaller and 15 lighter kernels than normal stocks, it has been pointed out that it would be unsafe to conclude that opaque-2 types have necessarily a lower yield than normal i80genic types. The data indic~te that modifier genes affect kernel size in opaque-2 homozygotes;
therefore appropriate selection in segregating populations should 20 be effective in improving that trait. Other researchers are of a similar opinion and point out ~hat the density of opaque-2 kernels is dependent on the genetic background which would mske it possible to select lines in which opaque-2 shows higher densities.
The opaque-2 gene has been incorporated into corn hybrids 25 which commonly had a lower yield than their normal hybrid counterparts. Recently, however, improvements in yield have spurred renewed interest in high lysine corn usage.
Although much effort has been expended to increase the lysine content of maize proteins, very little effort has gone 30 towards the increase of lysine content in maize by increasing the free pool Iysine content.
Plant regeneration from cells in culture i8 essential for the application of somatic hybridization, for the production of new varieties through somoclonal variation or in vitro selection, and for 35 the use of genetic engineering in producing new varieties.
Although plants can be regenerated from tissue culture of several lZ~Z9ti~
varieties of corn, there are many varieties for which this has not been accomplished using similar techniques.
In recent years, plant cell culture successes have had a considerable influence on the understanding of the respective roles 5 of cell and organism in control of plant growth and development.
Isolated plant cells have been shown to be amenable to ~n vitro culture and complete plants have been regenerated from cultures derived from somatic tissues, either directly via somatic embryogenesis or indirectly via organogenesis. Generally, the 10 regeneration pathway of choice is determined empirically by the manipulation of extrinsic factors, especially growth regulators.
Early investigations of certain plant species have ~uggested that exogenous auxin concentration is a major factor controlling somatic embryogenesis, such that its reduction leads to the initiation of 15 embryoid formation, In other species, expo~ure to a definite balance of auxin and cytokinin leads to the occurrence of organogenesi~ (shoots, then roots). Although several genotypes of corn have been regenerated using these techniques, no process i8 generally applicable to most genotypes of corn. Many genotypes 20 remain extremely difficult, if not impossible, to culture using the prior processes.
The process which has become the standard gystem for corn tissue culture i8 described by Green et al ., Crop Science 15 , 417 (1975). In this process, immature embryos were plated onto a 25 calluæ induction medium which comprises the MS mineral salts, Straus vitamins and amino acids ( glycine, asparagine, niacin, thiamine, pyridoxine and pantothenic acid), 2% sucrose, 0.8~6 agar and a hormone ~elected from 2, 4-dichlorophenoxyacetic acid (2, 4-D), p-chlorophenoxyacetic acid (p-CPA), -naphthaleneacetic 30 acid (NAA), 2isopentyladenine (2-ip) or mixtures thereof.
Hormone concentrations which were useful were 2 mg/l 2,4-D and a mixture of 1 mg/l 2, 4-D, 4 mg/l NAA and 0 . 05 mg/l 2-ip .
Plantlets were regenerated by subculturing the callus on medium containing reduced hormone concentrations. Regeneration was then 35 accomplished on medium containing 0.25 mg/l 2,4-D or a mixture of mg/l NAA and 0 . 05 mg/l 2-ip, re~pectively . ~ll culturing wa~
96~
conducted in a 16 hour light/8 hour dark cycle for 3-4 week intervals before transfer. This reference reports that callus induction did not occur in one of five genotypes tested.
Similar results with different n~3dia have been den~nstrated by Fr~eling et al., Maydica 21, 97 (1976); Vasil et al., eor.
Genet. 66, 285 (1983); E;dallo et al., Maydica 26,39 (1981); Lu et al., ~eor. ~p1. Genet. 62, 109 (1982); Gegenbach, Proc. Nat.
Acad. Sci. USA 74, 5113 (1977); and Green et al., Cr ~ Scienoe 14, 54 (1974). Ihe latter reference also den~nstrates genotype effects on ca11us inductian.
Although this procedure hss generally been unsuccessful for regenerating plants from al] maize genotypes, the regeneration of most genotypes is now possible through the substitution of dicamba for 2, 4-D in the media . See published European Application No .
0 177 738 snd Duncan et al.f Planta 165, ~22 (1985).
. _ SUMMARY OF THE IN~7ENTION
. .
The present invention is directed to a process for increasing the free pool lysine content in maize seed and for increasing the average free pool lysine content of seeds produced by a maize plant. The process comprises the regeneration of plants from tissue in culture which has been selected through the use of a Iysine anslog, More specifically, the present process comprises the steps of:
(a) culturing tissue obtained from a maize plant on a callus induction medium comprising mineral salts, vitamins, sucrose and a hormone to form callus;
(b) subculturing said callus on selection medium comprising mineral salts, vitamins, sucrose, S-2-aminoethyl-L-cysteine (AEC) snd a hormone to produce selected callus; snd (c) sub&ulturing said selected callus on regeneration medium comprising mineral salts, vitamins snd ~ucrose to regenerate plants.
Alternatively, the prscess can comprise the additional step of subculturing the callus maintenance medium comprising mineral lZ9Z960 salts, vitamins, sucrose and a hormone before subculturing on the selection medium.
The plants are gro~ n to produce seed (Rl) which can be used to produce further generations of plants.
The present invention is directed to a pro~es~ for increasing the free pool lysine content in maize seed and fo- increasing the average free pool lysine content of seeds produced by a maize plant. The process utilize~ tissue culture selectior of maize tissue 10 with AEC as described further below.
The present invention is directed to maize seeds which have a *ee pool Iysine content of at least about 500 1Jg per gram dry seed weight. Preferably, each seed has at least about 600 ~g of free pool lysine per gram dr~ seed welght. In the most preferred 15 embodiment, each seed has at least 700 ~g of free pool lyEine per gram dry seed weight.
The present invention is further directed tt maize plants, each of which produces seeds having an average free pool lysine content of at least about 325 ~g per gram dr~ seed weight.
- 20 Preferably, each plant produces seeds having an a~erage free pool lysine content of at least about 400 llg per gram d~y seed weight.
It is most preferred that the average free pool lysine content be at least about 500 l~g per gram dry seed weight. It s~.ould be evident that "average free pool lysine content" refers to the average of 25 the free pool lysine content of the individual seeds produced by the maize plant.
The initial regenerated plants (Ro~ are produ~ed from tissue culture generally by regenerating plants from 'dssue in culture which has been selected through the use of a ly~;ine analog, S-2-30 aminoethyl-~-cysteine (AEC). It is also possible to isolate plants which meet the desired characteristics without the use of an AEC
selection scheme. The following generations of plants can be produced by germinating the seed (Rl seed~ produced by the Ro plants. The Rl plants can be self-pollinated to produce R2 seeds.
lZ9Z960 Alternatively, the Rl plants can be incorporated into a breeding program to produce hybrids, inbreds, etc. Any generation of plants having the initial R1 seed as a parent can be used to develop hybrids or inbreds, etc.
For example, if possible, the regenerated plants or their progeny are self-pollinated. In addition, pollen obtained from the regenerated plants or their progeny is crossed to seed grown plants of agronomically important inbred lines. In some cases, pollen from plants of these inbred lines is used to pollinate regenerated plants or their progeny. The trait is geneticall~-characterized by evaluating the segregation of the trait in first-and later-generation progeny. The heritability and expression in plants of traits selected in tissue culture are of particular importance if the traits are to be commercially useful.
The commercial value of maize having a~ increased free pool Iysine content is greatest if many different hybrid combinations are available for sale. The farmer typically grows more than one kind of hybrid based on such differences as maturity, standability or other agronomic traits. Additionally, hybrids adapted to one part of the corn belt are not adapted to another part because of differences in such traits as maturity, disease and insect resistance. Because of this, it is necessary to breed increased free pool Iysine content into a large number of parental lines so that many hybrid combinations can be produced.
Adding the increased free pool Iysine genotype to agronomically elite lines is most efficiently accomplished if the genetic control of free pool lysine content is understood. This requires crossing increased free pool Iysine plants with low free pool ly~ine plants and studying the pattern of inheritance in segregating generations to ascertain whether the trait is expressed as a dominant or recessive, the number of genes involved, and any possible interaction between genes if more than one is required for expression. This geneffc analysis can be part of the initial efforts to convert agronomically elite, yet }ow free pool 3~ lysine content, lines to increflsed free pool lysine lines.
lZ~Z9f~0 A conversion process (backcrossing) is csrried out by crossing the original increased free pool lysine line to normal elite lines and crossing the progeny back to the normal parent. The progeny from this cross will segregate such that some plsnts carry 5 the gene(s) responsible for increased free pool lysine, whereas some do not. Plants carrying such genes will be crossed again to the normsl parent, resulting in progeny which segregate for increased free pool lysine ar.d normal production once more. This is repeated until the original normal parent has been converted to 10 sn increased free pool line, yet possesses all other importsnt attributes as originally found in the normsl parent. A separate backcrossing program is implemented for every elite line that is to be converted to an increased free pool lysine line.
Subsequent to the backcrossing, the new lines and the 15 appropriate combinations of lines which malce good commercial hybrids are evsluated for increased free pool lysine content as well as 8 battery of important agronomic traits, Increased free poo]
lysine lines and hybrids are produced which are true to type of the originsl normal lines and hybrids. This requires evalustion 20 under a range of environmental conditions where the lines or hybrids will generally be grown commercially. For production of increased free pool lysine maize, it may be necessary that both parents of the hybrid seed corn be bomozygous for the increased free pool lysine characterO Parental lines of hybrids that perform 25 satisfactorily are increased and used for hybrid producffon using standard hybrid seed corn production practices.
In general, the process to produce Ro plants comprises (a) culturing maize tissue on a medium to produce callus; (b) selecting callus tissue which is resistant to AEC in the culture medium, and 30 (c) regenerating plants from the ~elected callus. The callus can optionally be maintained on a maintenance medium prior to tran~fer to a selecffon medium. The regenerated plants are matured and self-pollinated to produce Rl seeds. R2 seeds are produced by self-pollinating Rl plants germinated from ~1 seeds. Additional 35 generations can be obtained in like manner.
lZ9Z9~iO
Although the process to increase free pool lysine content generally uses a selection scheme with AEC, it is possible to produce maize seeds and plants having the desired characteristics without selection. In this instance, the callus is maintained on a 5 maintenance medium rather than cultured on a selection medium.
This process is not as efficient , i . e ., it has a lower frequency of success than selection with AEC.
Within the general process outlined above, several tissue culture sequences can be utilized to produce maize seeds and 10 plants having the desired characteristics. The preferred sequences can be summarized as follows:
In the first preferred sequence, callus is induced on a callus induction mediu~ as described below. The callus is then transferred to maintenance medium for 6-9 passages before transfer 15 to regeneration medium. The plants are then transferred to soil.
No selection is performed àt any step during this sequence.
In a second preferred sequence, callus is induced on a callus induction medium as described below. The callus induction medium could contain 0-0.2 mM AEC. If no AEC is contained in the callus 20 induction medium, the callus is transferred to maintenance medium for 2-6 passages before it is transferred to selection medium. If the callus induction medium contains AEC, the callus is then transferred to selection medium rather than to maintenance medium.
The callus is transferred to selection medium for 3-8 passages 25 before transfer to regeneration medium. The concentration of AEC
i8 increased step~ise from 0 .1 to 3 . O mM, preferably from 0 .1 to 1.0 mM or from O.S to 2.0 mM, through the passages. In~an alter-native, the AEC concentration is dropped to zero after a stepwise increase in concentration, i.e., transfer to a maintenance medium, 30 and then raised to 1.5-2.5 mM, preferably 1.5-2.0 mM, in the next transfer before proceeding to regeneration medium. 2-4 passages, preferably 3 pas~ages, on regeneration medium may be performed prior to transferring the plants to the soil. The regeneration medium may contain 0-0.5 mM AEC. It is preferred to utilize AEC
35 in the regeneration medium and to lower its concentration stepwise through the passages on the regeneration medium.
lZ929~0 g The third preferred sequence is silailar to the second sequence except that the concentration of AEC is increased stepwise from 1. 0 to 3 . 0 mM, preferably from 1. 0 to 2 . 0 mM, during the passages on the selection medium.
In a fourth preferred sequence, callus is induced on a callus induction medium as described above. The callus induction medium could contain 0-0.2 mM AEC. If no AEC is contained in the callus induction medium, the callus is transferred to maintenance medium for 2-6 passages before it is transferred to selection medium. If the callus induction medium contains AEC, the callus is then transferred to selection medium. The callus i8 transferred to selection medium for 3-8 passages before transfer to regeneration medium . The concentration of AEC may be 0 . 25-0 . 5 mM for 1-3 passages, preferably 2 passages, and then it is raised to 2.0-3.0 mM, preferably 2 . 0 mM, for 1-2 passsges, preferably 1 passage.
The AEC concentration is then lowered to û.5 mM and increased stepwise to 1. 0-1. 5 mM over 3-6 passages, preferably 4 passages .
In an alternative, the AEC concentrat~on is dropped to zero after a stepwl8e increase in concentration, i.e., transfer to a maintenance medium, and then rai~ed to 1.5-2.0 mM in the next transfer before proceeding to regeneration medium. 2-4 passages, preferably 3 passages, on regeneration medium msy be performed prior to transferring the plants to the soil. The regeneration medium may contain 0-0 . 5 mM AEC . It is preferred to utilize AEC in the regeneration medium and to lower its concentration stepwise through the passages on the regeneration medium.
The preferred sequences described above can be modified in accordance with the teachings herein and within the skill of the art. Several modifications include (a) reduced number of transfers on certain of the media, (b) addition of other media to the -sequence, (c) use of other basal media well known in the art, (d) the deletion of the transfers to maintenance media, (e) addition of more traTufers to maintenance media du~ng the overall selection scheme, (f) reduced passage duration, (g) increased number of transfers on each medium, and (h) modification of AEC in sequence.
lZ929~0 The plant tissue which is preferred for use in iniffation of callus is the immature embryo. It is preferred to use an immature embryo from a plant w hich has an elevated level of lysine. Such maize lines include lines 1007, 1008, 1010 and 1012 of Crow's 5 Hybrid Corn Company, Milford, Illinois. The imm~ture embryos are isolated from the cob at approximately 10-17 da~s post-pollination when the embryos are 1.0-2.0 mm, preferabl~ mm, in length.
The cob is harvested and surface-sterilized. ~he immature embryos are isolated from each kernel. The embryos are plated onto callus 10 induction medium which may or may not contair; AEC. The embryos are plated so that the embryo axis is in contact with the medium, i.e., the scutellar side is up.
The callus induction medium comprises mineral salts, vitamins and sucrose. The mineral salts comprise macroelements and 15 microelements. The macroelements used in the callus induction medium may be the following compounds: magnesium sulfate, calcium chloride, monopotassium phosphate, potassium nitrate and ammoniùm sulfate. The microelements contained in the callus induction medium are: boric acid, manganese sul'ate, zinc sulfate, 20 potassiurn iodide, iron (Il) sulfate, disodiu=-ethylenediamine-tetracetic acid (EI)TA), sodium molybdate (VI), copper (II) sulfate and cobalt chloride. This combination of mineral calts is known in the art ~ the N6 mineral salts, which have been modified to contain mineral salts of copper, cobalt and mol~bdenum. Other 25 combinations of mineral salts may also be used as long as they do not adversely affect callus induction. Examples of combinations of mineral salts include, but are not limited to, ~S, Heller, Nitsch and Nitsch~ B5 and White.
The preferred amounts of the macroelements and microelements 30 used to prepare one liter of the callus induc~on medium are as follows: 185 mg magnesium sulfate heptahydrate, 166 mg calcium chloride dihydrate, 400 mg monopotassium phosphate, 2830 mg potassium nitrate, 463 mg ammonium sulfate, 1.6 llig boric acid, 4.4 mg manganese sulfate monohydrate, 1.5 mg rinc sulfate hepta-35 hydrate, 0.83 mg potassium iodide, 27.8 mg iron (II) sultateheptahydrate, 37.3 mg disodium-EDTA, 0.25 mg ~odium molybdate lZ9Z960 (VI) dihydrate, 0.025 mg copper (Il) ~ulfate pentahydrste and 0.0a5 mg cobalt chlo~qde hexahydrate.
The callus induction medium ~so contains vit~;nins. ~he vitamins used include myo-inositol, nicotinic acid, glycine, X pyridoxine, thiamine, and pantothenate. The amounts of ~ itamins used to prepare one liter of the callus induction medium are ~s follows: l00 mg myo-inositol, 0.5 mg nicotinic acid, 2 mg glycine, 0.5 mg pyrido~ne hydrochloride and ~.0 mg thiamir~e hydrochloride and 0.25 mg calcium pantothenate.
The first medium contains 2-6%, preferably 3%, ~ucrose and a gelling agent such as agar or GelriteTM (tr~demark, Kelco Commercial Development, P.O. Box 23076, San Diego, California).
It is preferred to use Bacto-Agar st a concentration of about 0.78%. The medium has a pH of 5.5-6.0, w;th a preferred pH of 5.8, before autocls~ring.
In sddition to the above components, the callus induction medium also contains a hormone. As used herein, "hormoner is lntended to mean any natural or synthetic compound which has a regulatory effect on plants or plant tissue. Plant hormones include auxins and cytokinins. The hormone which i~ useful for callus inducffon h the present in~.rention is 2,4-D. 2,4-D can be utilized alone or in combination with another hormone such as 2,4,5-trichlorophenoxyacetic scld (2,~,5-T) or zeatin. Other hormones, such ~ those described in publi~hed European application No, 0 177 738 and copending U.S. Patent No.
4, 830, 966, can be usea. The amount of honnone is ~ufficient to insure callus formation. Generally, about 2-3 mg/1 of 2, 4-D is sufficient, with 2 mg/1 preferred.
If zeatin or 2, 4, 5-T are also present, they can be utilized ln an amount of 2-3 mg/1, preferably about 2 mg/1.
The callu~ induction medium may also contain a low concentration of ~EC. It i8 preferred that no ~EC be pre~ent in this medium. If AEC i6 pre~ent, it i~ preferred that a concentratlon of 0.05-0.2 mM be utilized.
The immature embryos are plated on the callus induction medium and cultured in dif u~ed light wlth a photoperiod of 16 lZ'329~0 hours per day for 2-5 weeks, preferably 3-4 weeks. Dulqng this time, the embryo undergoes de-differentiation and callus formation.
After culturing the immature embryo on the callus induction medium, the callus is transferred and subcultured on either a maintenance medium or a selection medium. Generally, the only difference between the maintenance medium and the selection medium is that the latter contains AEC. The callus may be cultured on the maintenance medium for- 3-6 transfers before it is transferred to selection medium. The callus may be cultured on the selection medium for 3-8 transfers before it is transferred to regeneration medium. The callus is transferred to fresh maintenance or selection medium every 10-70 days.
The maintenance and selection media comprise mineral salts, vitamins, sucrose and a hormone in an amount sufficient to maintain the callus. The mineral salts and vitamins are as described for the callus induction medium. As in the callus induction medium, various combinations of mineral salt~ which do not adversely affect the functioning of the medium may be utilized.
The sucrose concentration is 3-6~, preferably 396. The hormone is generally 2,4-D at a concentration of 2-3 mg/l, preferably 2 mg/l, although others can be used as discussed above. Bacto-Agar at 0.78% is used to solidify the medium.
The maintenance and selection media may further contain L-glutamine and citric acid. If these components are utilized, it is preferred to use a concentration of 10 mM L-glutamine and 5 mM
citric acid.
The selection medium further contains AEC. The concentration of AEC in the selection medium may range from 0 .1-3 . 0 mM, preferaMy 0.1-2.0 mM. The concentration of AEC is varied during the selecffon process, as will be described further below.
After completing the selection sequence, the selected callus is placed on regeneration medium. One or more regeneraffon media may be utilized, with transfers to fresh regeneration medium occurring at about 5-40 days. Generally, 2-4 transfers may be Ufflized~
l;~9Z960 The regeneration medium comprises mineral salts, vitamins and sucrose. The mineral salts may be the same as for the callus induction medium, i.e., N6 salts, or they may be the MS mineral salts. It i~ preferred to utilize N6 salts.
The regenerstion medium also contains vitamins. The vitamins which may be utilized are either (a) thiamine, nicotinic acid, pyridoxine and glycine, or (b) thiamine, glycine and myo-inositol.
The (a) vitamins are referred to herein as N6 vitamins plus glycine, and the (b) vitamins are referred to herein as vitamin G.
The amount of vitamins used to prepare one liter of regeneration medium is as follows: (a) for N6 vitamins: 1 mg thiamine hydro-chloride, 0.5 mg nicotinic acid, 0.5 mg pyridoxine hydrochloride and 2 mg glycine; and (b) for vitamin G: 1 mg thiamine hydro-chloride, 2 mg glycine and 100 mg myo-inositol.
It is preferred that the regeneration medium contains 2-6%
sucrose, and the gelling substance may be Bacto-Agar or Gelrite at about 0.78% or about 0.2296, respectively.
The regeneration medium may contain AEC at a concentration of 0 .1-0 . 50 ml~ . It is preferred to reduce the AEC concentration during the transfers which are performed at about 5-40 days during the regeneration cycle. The regeneration medium may also contain a hormone. It i~ preferred to utilize one or more cytokinins if a hormone is used. The hormone may be selected from a mixture of (a) 0.2-0.5 mg/l indoleacetic acid (IAA) and 0 . 4-1 . 5 mg/l benzyl amino purine (BAP), preferably either 0 . 3 mg/l and 1.0 mg/l, respectively, or 0.3 mgll and 0.5 mg/l, respectively, or 0.25 mgll and 1.0 mgll, respectively; (b) 0.2-0.5 mgll IAA and 0.8-1.5 mg/l BAP, preferably 0.3 mg/l and 1.0 mgll, respectively; and (c) 0.1-0.3 mgll 2,4-D, 0.05-0.2 mg/l BAP
and 0.2-0.5 mgll gibberellic acid (GA3), preferably 0.2 mg/l, 0.1 mg/l and 0.35 mg/l, respectively.
After plants have been regenerated and established, they are transferred to cubes which contain one part of potting soil and one part of vermiculite. The gelling substance i8 washed off the plants prior to transfer to the cubes. The plantg are transferred to 12" pots containing potting 80il after about 4-40 days and 12929f~
. .
placed in the greenhouse. All of the culturing described above and that described below in the examples i8 conducted at about 24C with a 16 hour diffused light/8 hour dark cycle.
In several instances, the Rl seeds produced by plants obtained in accordance with the above process showed a decreased free pool lysine content. However, plants germinQted from these Rl seeds produced R2 seeds having an average free pool Iysine content of ~t least 325 lJg per gram dry ~eed weight. Thus, a lo~
free pool lysine content in Rl seed does not indicate that the fin~l 10 desired characteristics cannot be obtained. It demonstrates that ~everal generations of plants may be re~uired to obtain plants and seeds having the desired characteristics.
The present invention will be further described by reference to the following non-limiting examples.
Preparation o~ Solutions The following Atock solutions or solutions were prepared for use in making the media described in further detail below.
1. OPA Reaction Mi~c OPA reaction mlx was prepared b~ dissolving 2.6 g of boric acid in ~0 ml of deionized water. The pH was adjusted to 10.4 with a 45~ golution of KOH~ 200 1ll of 2-mercaptoethanol was added, followed by 300 pl of Brij-35. Brij-3~ ~as added to gtabilize the OPA-lysine complex (Anal.Biochem. 101, 61 (1980)). 80 mg of 25 Fluoropa *(OPh from Pierce) was dissol~ed in I ml methanol and then added to the borate solution. The volume was sdjusted to 100 ml with deionized water. The OPA resction mix was kept at 4C and was di~carded after two weelcs to insure low background peaks.
* Trademark 2. AEC Stock Solution A 500 mM stock solution of AEC was prepared by dissolving 5 . 02 g of AEC in 500 ml of deionized water or by dissolving 1.004 g of AEC in 100 ml of deionized water. The pH was adjusted to 5.8 using lN KOH.
3. Hormones A. 2,4-D. A 0.5 mg/ml stock solutior. was prepared by dissolving 50 mg of 2,4-D in 100 ml of deionized water.
B. IAA. A 0.5 mg/ml stock solution was prepared by dissolving 50 mg of IAA in 100 ml of deionized ~ater.
C. BAP. A 0.5 mg/ml stock soluffon was prepared by dissolving 50 mg of BAP in 100 ml of deionize~ ~-ater.
D. Zeatin. A O.1 mg/ml stock solutior. was prepared by dissolving 10 mg of zeatin in 100 ml of deionizec w~ter.
E. aA3. A 0.167 mg/ml 8tock solution was prepared by di~solving 16,7 mg of GA3 in 100 ml of deionized water F . 2, 4, 5-T . A O . 5 mg/ml ~tock solution was prepared by dissolving 50 mg of 2,4,5-T in 100 ml of deioniz~d water.
.
B. IAA. A 0.5 mg/ml stock solution was prepared by dissolving 50 mg of IAA in 100 ml of deionized ~ater.
C. BAP. A 0.5 mg/ml stock soluffon was prepared by dissolving 50 mg of BAP in 100 ml of deionize~ ~-ater.
D. Zeatin. A O.1 mg/ml stock solutior. was prepared by dissolving 10 mg of zeatin in 100 ml of deionizec w~ter.
E. aA3. A 0.167 mg/ml 8tock solution was prepared by di~solving 16,7 mg of GA3 in 100 ml of deionized water F . 2, 4, 5-T . A O . 5 mg/ml ~tock solution was prepared by dissolving 50 mg of 2,4,5-T in 100 ml of deioniz~d water.
.
4. Vitamins A. N6 Vitamins. A lOOOX stock solution of N6 vitamins was prepared by dissolving 100 mg of thiamine hydrochloride, 50 mg of nicotinic acid, 50 mg of pyrido~dne hydrochloride and 200 mg of glycine in 100 ml of deionized water.
B. Vitamin G. A 1000X stock solution of vitamin G was prepared by dissolving 100 mg of thiamine hydrochloride, 200 mg of glycine and 10 g of myo-inositol in 100 ml of deionized water.
~; ~ C. Vitamins. A 1000X stock solution of vitamins was prepared by dissolving 100 mg of thiamine hydrochloride, 50 mg of pyridoxine hydrochloride, 50 mg of nicotinic acid, 200 mg oi - 129Z9~0 glycine, lO g of myo-inositol and 25 mg of calcium pantothenate in 100 ml of deionized water. The pH was adjusted to 5. 8 using lN
XOH .
B. Vitamin G. A 1000X stock solution of vitamin G was prepared by dissolving 100 mg of thiamine hydrochloride, 200 mg of glycine and 10 g of myo-inositol in 100 ml of deionized water.
~; ~ C. Vitamins. A 1000X stock solution of vitamins was prepared by dissolving 100 mg of thiamine hydrochloride, 50 mg of pyridoxine hydrochloride, 50 mg of nicotinic acid, 200 mg oi - 129Z9~0 glycine, lO g of myo-inositol and 25 mg of calcium pantothenate in 100 ml of deionized water. The pH was adjusted to 5. 8 using lN
XOH .
5. Amino Acids A. Glutamine. A 250 mM stock solution was prepared by dissolving 3.65 g of L-glutamine in lOO ml of deionized water. Ihe pH was adjusted to 5.8 with lN KOH.
B. Citric Acid. A 1 M stock solution was prepared by dissolving 19 . 2 g of anhydrous citric acid in 100 ml of deionized water. The pH was adjusted to 5.8 with lN KOH.
B. Citric Acid. A 1 M stock solution was prepared by dissolving 19 . 2 g of anhydrous citric acid in 100 ml of deionized water. The pH was adjusted to 5.8 with lN KOH.
6. Mineral Salts A. Modified N6. A 20X stock solution of modified N6 salts was prepared by d~ssol~ng 1850 mg of magnesium sulfate heptahydrate, 1660 mg of calcium chloride dihydrate, 4 . O g of monopotassium phosphate, 4 . 63 g of ammonium sulfate, 28 . 3 g of potassium nitrate, 16 m~ of boric acid, 44 mg of manganese sulfate - monohydrate, 15 mg of zinc sulfate heptahydrate, 8 mg of potassium iodide, 278 mg of iron (II) sulfate heptahydrate, 373 mg of disodium-EDTAf 2.5 mg of sodium molybdate (VI) dihydrate, 0.25 mg of copper (II) sulfate pentahydrate and 0.25 mg of cobalt chloride hexahydrate in 300 ml of deionized water. The volume was then brought to 500 ml and divided into 50 ml aliquots.
B. MS. A 20X stock solution of MS salts was prepared by dissoIving lO packages of MS salts, unbuffered (Gibco Catalog ~o.
600-1117) in 500 ml of deionized water. The stock solution was divided into 50 ml aliquots.
: :~
Preparation of Media 1, 2D/Z3S. This medium wss prepared by adding 30 g of sucrose, 50 ml of 20X modified N6 6alts and 4 ml of 2, 4-D stock 601ution to 600 ml of deionized water. The volume was brought up to ~79 ml with deionized water. The pH was adju~te~ to 5.8 with lN KOH, 7.8 g of Bacto-Agar were added, and the mixture was autoclaved. 20 ml of zeatin ~tock solution and 1 ml of ~itamins stock solution were filter-sterilized and added to the cooling medium, which was poured into petri dishes.
2. IOT3S. This medium was prepared by adding 30 g of sucrose, 50 ml of 20X modified N6 salts, 4 ml of 2,4-D 6tock solution and 4.4 ml of 2,4,5-T ~tock ~olution to 600 llil of deionized water. The volume was brought up to 999 ml with deionized water and the pH adjusted to S . 8 with lN KOH . 7 . 8 g of Bacto-Agsr were added and the mixture autocla~ved. 1 ml o lritsmins 6tock ~olution ws~ ~ilter-~terilized and added to the cooling medium, which was poured into petri di~hes.
3. 3NM. This medium was prep~red by adding 30 g of sucror,e, SO ml of 20X modified X6 ~alts and 4 ml of 2,4-D stock ~olution to 600 ml of deionized water. Addiffonal deionized water was added to bring the volume to ~54 ml. The pH was adjusted to 5.8 using lN KOH and 7.8 g of Bacto-Agar were added. The mixture was autoclaved for 20 minutes at 20 psi and 250F. 40 ml of the glutamine stocX solution, 5 ml of the citric acid stock solution, ~d 1 ml of the vltsmin~ stock solution were each sterilized by flltration through a 0.22 micron Millipore*membrane or a 0.2 micron Gelmsn*filter and then added to the cooling medium, which was poured into petri dishes.
,: , ~. AO.2N. This medium wss prepsred as described for 3NM
except that the volume was brought up to ~50 ml before ~utocls~rlng. 4 ml of the ~C stock solution were ~ter-~terilized * Trademark ~ .
and added to the cooling medium along with the glutamine, citric acid snd vitamins.
5 . AO . 25N . This medium was prepared as described above except that the volume was brought up to 949 ml before 5 autoclaving and 5 ml of AEC stock solution were used.
6. AO.5N. This medium was prepared as described above except that the volume was brought up to 944 ml before - autoclaving and 10 ml of AEC stock solution were used.
B. MS. A 20X stock solution of MS salts was prepared by dissoIving lO packages of MS salts, unbuffered (Gibco Catalog ~o.
600-1117) in 500 ml of deionized water. The stock solution was divided into 50 ml aliquots.
: :~
Preparation of Media 1, 2D/Z3S. This medium wss prepared by adding 30 g of sucrose, 50 ml of 20X modified N6 6alts and 4 ml of 2, 4-D stock 601ution to 600 ml of deionized water. The volume was brought up to ~79 ml with deionized water. The pH was adju~te~ to 5.8 with lN KOH, 7.8 g of Bacto-Agar were added, and the mixture was autoclaved. 20 ml of zeatin ~tock solution and 1 ml of ~itamins stock solution were filter-sterilized and added to the cooling medium, which was poured into petri dishes.
2. IOT3S. This medium was prepared by adding 30 g of sucrose, 50 ml of 20X modified N6 salts, 4 ml of 2,4-D 6tock solution and 4.4 ml of 2,4,5-T ~tock ~olution to 600 llil of deionized water. The volume was brought up to 999 ml with deionized water and the pH adjusted to S . 8 with lN KOH . 7 . 8 g of Bacto-Agsr were added and the mixture autocla~ved. 1 ml o lritsmins 6tock ~olution ws~ ~ilter-~terilized and added to the cooling medium, which was poured into petri di~hes.
3. 3NM. This medium was prep~red by adding 30 g of sucror,e, SO ml of 20X modified X6 ~alts and 4 ml of 2,4-D stock ~olution to 600 ml of deionized water. Addiffonal deionized water was added to bring the volume to ~54 ml. The pH was adjusted to 5.8 using lN KOH and 7.8 g of Bacto-Agar were added. The mixture was autoclaved for 20 minutes at 20 psi and 250F. 40 ml of the glutamine stocX solution, 5 ml of the citric acid stock solution, ~d 1 ml of the vltsmin~ stock solution were each sterilized by flltration through a 0.22 micron Millipore*membrane or a 0.2 micron Gelmsn*filter and then added to the cooling medium, which was poured into petri dishes.
,: , ~. AO.2N. This medium wss prepsred as described for 3NM
except that the volume was brought up to ~50 ml before ~utocls~rlng. 4 ml of the ~C stock solution were ~ter-~terilized * Trademark ~ .
and added to the cooling medium along with the glutamine, citric acid snd vitamins.
5 . AO . 25N . This medium was prepared as described above except that the volume was brought up to 949 ml before 5 autoclaving and 5 ml of AEC stock solution were used.
6. AO.5N. This medium was prepared as described above except that the volume was brought up to 944 ml before - autoclaving and 10 ml of AEC stock solution were used.
7. AlN. This medium was prepared as described above 10 except that the volume was brought up to 934 ml before autoclaving and 20 ml of AEC stock solution were used.
8. A2N. This medium was prepared 8S described above except that the volume was brought up to 914 ml before autoclaving and 40 ml of AEC stock solution were used.
lS 9 . AO . O5N . Thi~ medium was prepared as described for AO . 2N except thst 1 ml of AEC stock solution was used and the initisl volume brought up to 953 ml.
10, AO. 1~ . Thi~ medium was prepared as described for AO . 2N except that 2 ml of AEC stock solution were used and the 20 initial volume brought up to 952 ml.
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11. 2N3. This medium was prepared by adding 30 g of sucrose, 50 ml of 20X modified N6 sslts and 4 ml of 2,4-D stock solution to 600 ml of deionized water. Addiffonal deionized water wss added to bring the volume to 999 ml. The pH was adjusted to 25 5,8 using 1~' KOH, and 7.8 g of Bacto-Agar were added. The mixture was autoclaved as previously described, 1 ml of the vitamins stock solution was filter-sterilized and added to the cooling medium, which was poured into petri dishes.
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12. 2N6. This medium was prepared as described for 2N3 except that 60 g of sucrose were added instead of 30 g.
13. 3N3. This medium was prepared as described for 2N3 except that 6 ml of 2, 4-D stock solution were added instead of 5 4 ml.
14. 3N. This medium was prepared by adding 30 g of sucrose, 50 ml of 20X modified N6 salts and 4 ml of 2, 4-D stock solution to 600 ml of deionized water. Additional deionized water was added to bring the volume up to 954 ml. The pH was adjusted 10 to 5 . 8 with lN KOH and 7 . 7 g of Bacto-Agar were added . The mixture was autoclaved. 40 ml of glutamine stock solution, 5 ml of citric acid stock solution and 1 ml of vitamins stock solution were filter-sterilized and added to the cooling medium. The medium was poured into petri dishes.
15. MAC15, This medium was prepared as described for 2N3 except that the volume was brought up to 969 ml before autoclaving. 30 ml of AEC stock solution were filter-sterilized and added to the coolhg medium along with the vitamins. The medium was then poured into petri dishes.
16, MAC20. This medium was prepared as described for MAC15 except that the volume was brought to 959 ml before autoclaving and 40 ml of AEC stock solution was used.
17. MAC10. This medium was prepared as described for MACI5 except that the volume before autoclaving was 979 ml and 20 ml of AEC stock solution we~e used instead of 30 ml.
18 . RMl . 5 . This medium was prepared by adding 00 g of sucrose, 50 ml of 20X modified N6 salts and 1 ml of vitamin G
stock solution to 600 ml of deionized water. The volume was brought up to 990 ml by the addition of deionized water. The pH
was ad~usted to 5 . 8 with lN KOH, and 2 . 2 g of Gelrite were added. The mixture was autoclaved. 10 ml of AEC stock solution was filter-sterilized and added to the cooling medium. The mixture was then poured into petri dishes.
19. RM2 . 25 . This medium was prepared by adding 20 g of sucrose, 50 ml of 20X modified N6 salts, O. 6 ml of IAA stock solution, 2.0 ml of BAP stock solution, and 1 ml of vitamin G stock solution to 600 ml o~ deionized water. The volume was brought up to 995 ml by the addition of deionized ~ater. The pH was adjusted to 5 . 8 with 1~ KOH, and 2 . 2 g o' Gelrite were added .
The mixture was autoclaved, and 5 ml of AEC stock solution was filter-sterilized and added to the cooling medium. The mixture was then poured into petri dishes.
20. RM(2). ~his medium was prep~-ed as described for RM2 . 25 except that the volume was brou g}: to I I and AEC was not added.
21. RM1. 25, This medium was prep~-ed as described for RM1. 5 except that the volume was broug-.t to 9g5 ml before autoclaving and 5 ml of AEC stock solutior. ~-ere used instead of 10 ml.
22. HV12A. 'rhis medium was prepared by adding 20 g of sucrose, 1 package of Gibco MS salt~ (Catalog No. 500-117), 0.4 ml of 2,4-D stock solution and 0.2 ml of 8AP stock solution to 600 ml of deionized water. The volume was brought up to 9g2 . 9 ml with the addition of deionized water. The pE~ W8S adjusted to 5.8 with lN KOH and 2 g of Gelrite were added. The mixture was autoclaved . 5 ml of AEC stock solution and 2 . I ml of GA3 stock solution were each f;lter-sterilized and added to the cooling medium. The mixture was then poured into petri dishes.
23. RRM. This medium was prepared by adding 15 g of sucrose, 50 ml of 20X MS salts, 1 ml of vita:Din G stock solution and 0.2 ml of 2,4-D stock solution to 600 ml o~ deionized water.
~29Z960 The volume was brought up to l 1 by the addition of deionized water, and the pH ad~usted to 5 . 8 with lN KOH . 8 . 5 g of Bacto-Ag~r were added. The mixture was autoclaved and poured into plant and tissue cult~re containers ( Flow general Company Catalog No. 26-721-07).
24. RRMl. This medium was prepared as described for RRM
except that the volume was brought up to 998 ml. 2 ml of AEC
stock solution were filter-sterilized and added to the cooling medium before it was poured.
25. IBM. This medium was prepared by adding 30 g of sucrose, 50 ml of 20X MS salts, 1 ml of vitamin G stock solution, 0.6 ml of IAA stock solution and 2 ml of BAP stock solution to 600 ml of deionized water. Deionized water was added to bring the volume to 1 1. Th,e pH was fldjusted to 5.8 with lN KOH, and 8.3 g of Bacto-Agar were added. The mixture was heated to dissolve the agar, and poured into te8t tubes 80 that the test tubes were one-third full. The tubes were then autoclaved as described above.
26. IBM7. This medium was prepared as described for IBM
except that l ml of BAP stock solution were used instead of 2 ml of BAP stock solution.
' 27. IBM12. This medium was prepared as described for IBM
except that 0 . 5 ml of IAA stock solution was used instead of 0,6 ml.
Immature 13mbryo Isolation Immature embryos were isolated from the cob of corn from lines 1007, l008, 10l0 or 1012 of Crow's Hybrid Corn Company, Milford, Illinois, 10-17 days post-pollination, when they were 1-2 30 mm, preferably l.5 mm, in length. The cob wa~ harvested and 125~Z9~0 surface-sterilized in a 20% 501ution of bleach and 1 drop of Liquinox~ (Alconox Inc., 853 Broadway, New York, N.Y.) detergent for 20 minutes. The cobs were rinsed with sterile, deionized water. The immature embryos were isolated by ~licing 5 off the top of each kernel with a scalpel and scooping out the endosperm. The immature embryos were then taken out and plated onto the desired medium, as described below, so that the embryo axis was in contact with the medium , i. e ., the scutellar side was up .
Basic Culturing Conditions and Corn Growth Immature embryos isolated as described above were plated onto a medium in a petri dish for the initiation of callus 80 that the embryo axis was in contact with the medium, i . e ., the 1~ scutellar side was up. Various media were ufflized for callus initiation, including 2D/Z3S, 10T3S, A0.05N, A0.2~, 3N and 2N6.
All culturing was conducted with a 16 hour diffused light/8 hour dark cycle at about 24C. The immature embryo ~as cultured for about 21 to about 32 days before transferring to fresh medium.
20 Transfers to fresh medium were usua]]y performed after about 6 to about 67 days, Various sequences of media ~-ere utilized as described in detail below. Generally, the callus was selected for a period of time on medium containing AEC, and then plants were regenerated. Regeneration was performed using several 2~ regenerating media, including RMl.5, RM2.25, RM1.25, RM(2), RR~, IBM12, IBM7, RRM1 and HV12A. A sequence of one or more of these media were utilized as described in further aetail below.
After plants were obtained, they were transferred to soil contained in cubes. The soil was comprised of a 1:1 mixture of vermiculite 30 and potffng 80il. After about 4 to about 30 days, each plant was transferred to a 12" pot containing standard nursery potting soil and placed in the greenhouse.
The plants were grown in the greenhouse and ~elf-pollinated.
The Rl seeds were collected and asssyed for free pool lysine l;~9Z9~0 content. Rl seeds were also planted in a field research nurser~-.
The Rl plants were self-pollinated and R2 seeds collected.
Culture Sequences Immature embryos isolated as previously described were cultured and plants regenerated as follows. All culturing wa~
conducted with a 16 hour diffused light/8 hour dark cycle.
1. Line 1012 (Tissue Culture Control) Immature embryos were plated on 3N medium. The callus was 1~ transferred and plants regenerated by the following sequence: to 3N medium after 26 days, to 3N medium after 67 days, to 3~1 medium after 21 days, to 3NM medium aftér 32 days, to 31~' medium after 28 days, to 3NM medium after 2~ days, to 31~' medium after 28 day~, to RM(2~ medium after 29 days, to cubes after 18 days, and to 12" pots after 29 days, One plant was identified as CZ0C22C~. The Rl seeds from this plant were assayed for free pool lysine 2, Line 1007 . . _ Immature embryos were plated on A0, 2N medium . The call~
was transferred and plants regenerated by the following sequence:
to A0.25N ~ medium after 21 days, to A0.5N medium after 25 dayc.
to AlN medium after 30 days, to AlN medium after 25 days, tc HV12A medium after 22 days, to IBM7 medium after 7 days, to RRM medium ~fter 23 days, to cubes after 53 days, and to 12-~ ~ 25 pots after 13 day~. One plant was identified as CZ1418A6, and the ;~ R1 seeds were assayed for free pool Iysine.
12929~
3. Line 1012 lmmature embryos were plated on 3N medium. The callus was transferred and plants regenerated by the following sequence: to 3N medium after 31 days, to 3N medium after 32 days, to 3N
medium after 35 days, to A0.25N medium after 19 days, to A0.25N
medium after 33 days, to A0 . 5N medium after 28 days, to A0 . SN
medium after 29 days, to AlN medium after 29 days, to AlN
medium after 31 days, to AlN medium after 11 days, to R?.11.25 medium after 19 days, to RM2.25 medium after 9 days, to RRM
medium after 29 days, to cubes after 12 days, and to 12" pots after 7 days. One plant was identified as CZ9D15Cl-3.
4. Line 1012 Immature embryos were plated on 3N medium. The callus was transferred and plants regenerated by the following sequence: to 3N medium after 28 days, to 3N medlum after 28 day8~ to 3N3 medium after 30 days, to 3N medium after 37 days, to A0 . 25N
medium after 19 days, to A0.25N medium after 33 days, to A0.5N
medium after 28 days, to A0.5N medium after 29 days, to AlN
medium after 24 days, to AlN medium after 26 days, to AlN
medium after 21 days, to RMl.25 medium after 19 days, to R~2.25 medium after 9 days, to IBM12 medium after 6 days, to RRM
medium after 13 days, to cubes after 23 days, and to 12r pots after 13 day6. One plant wPe identified as CZ9A3BlB9. A second plant identified as CZ9A3BlBll was produced by the same sequence. Rl seeds from these two plants were assayed for free pool ly~e. A third plant was identified as CZ9A3BlA6. The R1 seeds from the third plant were grown and the R2 seeds were analyzed for free pool lysine.
5. Line 1012 ., . . _ Immature embryos were plated on 3N medium. The callus was transferred and plants regenerated by the following sequence; to 1~9Z9~O
3N medium after 26 days, to 3N mediuc: after 30 days, to 3N
medium after 37 days, to A0.25N medium after 19 days, to A0.25N
medium after 33 days, to A2N medium sfter 21 days, to A0.5N
medium after 33 dsys, to A0.5N medium after 16 days, to AlN
medium after 25 days, to AlN medium after 33 days, to RMl.25 medium after 19 da~s, to RM2.25 medium after 9 days, to RRM
medium after 16 days, to cubes after 12 days, and to 12" pots after 22 days. Seven different plants were identified as:
CZ9B9AlAl, CZ9B9AlA6, CZ9B9AlB5, CZ9B9AlB30, CZ9A3A4-3, CZ9A3A4-6, and CZ9C27C2A4. The Rl s~ds from these plants were analyzed for free pool lysine. An additional plant identified as CZ9C27C2A17 was used to produce R2 seeds which were assayed for free pool lysine.
.
6. Line 1010 Immature embr~os were plated on 2N6 ~edium. The callus was trQnsferred and plants regenerated by the followlng sequence: to 2N6 medium after 32 days, to 2N3 mediun~ after 2~ days, to 3NM
medium after 11 d~ys, to 3NM medium after 27 days, to A0.5N
medium after 33 days, to A0.5N medium after 41 days, to AlN
20medium after 28 days, to AlN medium after 26 days, to MAC20 medium after 51 d~s, to MAC15 medium after 32 days, to RMl.5 medium after 44 days, to RM2 . 25 medium after 7 days, to RRMl medium after 33 days, to cube~ after 32 days, and to 12" pots after 4 days. Tv.-o individual plants were identified as CZllC8A2D
25and CZ11C8A2E. Rl seeds from these plants were assayed for free pool lysine.
7. Line 1010 Immature embryos were plated on 2D/Z35 medium. The callus was transferred and plants regenerated by the following sequence:
30to 2N6 medium after 32 days, to 2N3 medium after 27 days, to 3NM
medium after 11 days, to 3NM medium after 27 days, to A0.5N
medium after 33 days, to A0.5N medium after 41 days, to AlN
lZ9Z960 medium after 29 days, to Al~ medium after 23 days, to AlN
medium after 26 days, to MAC20 medium after 51 days, to 2N3 medium after 30 days, to MAC15 medium after 32 days, to RMl.25 medium after 44 days, to RM2.25 medium after 7 days, to cubes after 33 days, and to 12" pots after 22 days. Four different plants were identified as CZllC19AlAlB, CZllC19B2A7, CZllC19B2A8 and CZllC19B2A10. The Rl seeds were analyzed for free pooI Iysine.
8. Line lOlO
Immature embryos were plated on either lOT3S medium (CZllC18) or 2D/Z3S medium (CZllCl9). The callus was transferred and plants regenerated by the following sequence: to 2N6 medium after 32 days, to 2N3 medium after 27 days, to 3NM
medium after 11 days, to 3NM medium after 27 days, to A0.5N
15 medium after 33 d8ys, to A0.5N medium after 41 days, to AlN
medium after 29 days, to A0.5N medium after 23 days, to A0.5N
medium after 26 days, to MAC10 medium after 51 dsys, to 2N3 medium after 30 days, to MAC15 medium after 32 days, to RMl .5 medium after 44 days, to RM2.25 medium after 7 days, to RRMl 20 medium after 33 days, to cubes after 22 days, and to 12" pots after 4 days. Five plants were identified as CZllC18BlCl, CZllC18B2Cl, CZllC19AlBlB, CZllC19AlBlC and CZllC19AlB2B.
Rl seeds were assayed for free pool lysine.
lS 9 . AO . O5N . Thi~ medium was prepared as described for AO . 2N except thst 1 ml of AEC stock solution was used and the initisl volume brought up to 953 ml.
10, AO. 1~ . Thi~ medium was prepared as described for AO . 2N except that 2 ml of AEC stock solution were used and the 20 initial volume brought up to 952 ml.
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11. 2N3. This medium was prepared by adding 30 g of sucrose, 50 ml of 20X modified N6 sslts and 4 ml of 2,4-D stock solution to 600 ml of deionized water. Addiffonal deionized water wss added to bring the volume to 999 ml. The pH was adjusted to 25 5,8 using 1~' KOH, and 7.8 g of Bacto-Agar were added. The mixture was autoclaved as previously described, 1 ml of the vitamins stock solution was filter-sterilized and added to the cooling medium, which was poured into petri dishes.
129Z~O
12. 2N6. This medium was prepared as described for 2N3 except that 60 g of sucrose were added instead of 30 g.
13. 3N3. This medium was prepared as described for 2N3 except that 6 ml of 2, 4-D stock solution were added instead of 5 4 ml.
14. 3N. This medium was prepared by adding 30 g of sucrose, 50 ml of 20X modified N6 salts and 4 ml of 2, 4-D stock solution to 600 ml of deionized water. Additional deionized water was added to bring the volume up to 954 ml. The pH was adjusted 10 to 5 . 8 with lN KOH and 7 . 7 g of Bacto-Agar were added . The mixture was autoclaved. 40 ml of glutamine stock solution, 5 ml of citric acid stock solution and 1 ml of vitamins stock solution were filter-sterilized and added to the cooling medium. The medium was poured into petri dishes.
15. MAC15, This medium was prepared as described for 2N3 except that the volume was brought up to 969 ml before autoclaving. 30 ml of AEC stock solution were filter-sterilized and added to the coolhg medium along with the vitamins. The medium was then poured into petri dishes.
16, MAC20. This medium was prepared as described for MAC15 except that the volume was brought to 959 ml before autoclaving and 40 ml of AEC stock solution was used.
17. MAC10. This medium was prepared as described for MACI5 except that the volume before autoclaving was 979 ml and 20 ml of AEC stock solution we~e used instead of 30 ml.
18 . RMl . 5 . This medium was prepared by adding 00 g of sucrose, 50 ml of 20X modified N6 salts and 1 ml of vitamin G
stock solution to 600 ml of deionized water. The volume was brought up to 990 ml by the addition of deionized water. The pH
was ad~usted to 5 . 8 with lN KOH, and 2 . 2 g of Gelrite were added. The mixture was autoclaved. 10 ml of AEC stock solution was filter-sterilized and added to the cooling medium. The mixture was then poured into petri dishes.
19. RM2 . 25 . This medium was prepared by adding 20 g of sucrose, 50 ml of 20X modified N6 salts, O. 6 ml of IAA stock solution, 2.0 ml of BAP stock solution, and 1 ml of vitamin G stock solution to 600 ml o~ deionized water. The volume was brought up to 995 ml by the addition of deionized ~ater. The pH was adjusted to 5 . 8 with 1~ KOH, and 2 . 2 g o' Gelrite were added .
The mixture was autoclaved, and 5 ml of AEC stock solution was filter-sterilized and added to the cooling medium. The mixture was then poured into petri dishes.
20. RM(2). ~his medium was prep~-ed as described for RM2 . 25 except that the volume was brou g}: to I I and AEC was not added.
21. RM1. 25, This medium was prep~-ed as described for RM1. 5 except that the volume was broug-.t to 9g5 ml before autoclaving and 5 ml of AEC stock solutior. ~-ere used instead of 10 ml.
22. HV12A. 'rhis medium was prepared by adding 20 g of sucrose, 1 package of Gibco MS salt~ (Catalog No. 500-117), 0.4 ml of 2,4-D stock solution and 0.2 ml of 8AP stock solution to 600 ml of deionized water. The volume was brought up to 9g2 . 9 ml with the addition of deionized water. The pE~ W8S adjusted to 5.8 with lN KOH and 2 g of Gelrite were added. The mixture was autoclaved . 5 ml of AEC stock solution and 2 . I ml of GA3 stock solution were each f;lter-sterilized and added to the cooling medium. The mixture was then poured into petri dishes.
23. RRM. This medium was prepared by adding 15 g of sucrose, 50 ml of 20X MS salts, 1 ml of vita:Din G stock solution and 0.2 ml of 2,4-D stock solution to 600 ml o~ deionized water.
~29Z960 The volume was brought up to l 1 by the addition of deionized water, and the pH ad~usted to 5 . 8 with lN KOH . 8 . 5 g of Bacto-Ag~r were added. The mixture was autoclaved and poured into plant and tissue cult~re containers ( Flow general Company Catalog No. 26-721-07).
24. RRMl. This medium was prepared as described for RRM
except that the volume was brought up to 998 ml. 2 ml of AEC
stock solution were filter-sterilized and added to the cooling medium before it was poured.
25. IBM. This medium was prepared by adding 30 g of sucrose, 50 ml of 20X MS salts, 1 ml of vitamin G stock solution, 0.6 ml of IAA stock solution and 2 ml of BAP stock solution to 600 ml of deionized water. Deionized water was added to bring the volume to 1 1. Th,e pH was fldjusted to 5.8 with lN KOH, and 8.3 g of Bacto-Agar were added. The mixture was heated to dissolve the agar, and poured into te8t tubes 80 that the test tubes were one-third full. The tubes were then autoclaved as described above.
26. IBM7. This medium was prepared as described for IBM
except that l ml of BAP stock solution were used instead of 2 ml of BAP stock solution.
' 27. IBM12. This medium was prepared as described for IBM
except that 0 . 5 ml of IAA stock solution was used instead of 0,6 ml.
Immature 13mbryo Isolation Immature embryos were isolated from the cob of corn from lines 1007, l008, 10l0 or 1012 of Crow's Hybrid Corn Company, Milford, Illinois, 10-17 days post-pollination, when they were 1-2 30 mm, preferably l.5 mm, in length. The cob wa~ harvested and 125~Z9~0 surface-sterilized in a 20% 501ution of bleach and 1 drop of Liquinox~ (Alconox Inc., 853 Broadway, New York, N.Y.) detergent for 20 minutes. The cobs were rinsed with sterile, deionized water. The immature embryos were isolated by ~licing 5 off the top of each kernel with a scalpel and scooping out the endosperm. The immature embryos were then taken out and plated onto the desired medium, as described below, so that the embryo axis was in contact with the medium , i. e ., the scutellar side was up .
Basic Culturing Conditions and Corn Growth Immature embryos isolated as described above were plated onto a medium in a petri dish for the initiation of callus 80 that the embryo axis was in contact with the medium, i . e ., the 1~ scutellar side was up. Various media were ufflized for callus initiation, including 2D/Z3S, 10T3S, A0.05N, A0.2~, 3N and 2N6.
All culturing was conducted with a 16 hour diffused light/8 hour dark cycle at about 24C. The immature embryo ~as cultured for about 21 to about 32 days before transferring to fresh medium.
20 Transfers to fresh medium were usua]]y performed after about 6 to about 67 days, Various sequences of media ~-ere utilized as described in detail below. Generally, the callus was selected for a period of time on medium containing AEC, and then plants were regenerated. Regeneration was performed using several 2~ regenerating media, including RMl.5, RM2.25, RM1.25, RM(2), RR~, IBM12, IBM7, RRM1 and HV12A. A sequence of one or more of these media were utilized as described in further aetail below.
After plants were obtained, they were transferred to soil contained in cubes. The soil was comprised of a 1:1 mixture of vermiculite 30 and potffng 80il. After about 4 to about 30 days, each plant was transferred to a 12" pot containing standard nursery potting soil and placed in the greenhouse.
The plants were grown in the greenhouse and ~elf-pollinated.
The Rl seeds were collected and asssyed for free pool lysine l;~9Z9~0 content. Rl seeds were also planted in a field research nurser~-.
The Rl plants were self-pollinated and R2 seeds collected.
Culture Sequences Immature embryos isolated as previously described were cultured and plants regenerated as follows. All culturing wa~
conducted with a 16 hour diffused light/8 hour dark cycle.
1. Line 1012 (Tissue Culture Control) Immature embryos were plated on 3N medium. The callus was 1~ transferred and plants regenerated by the following sequence: to 3N medium after 26 days, to 3N medium after 67 days, to 3~1 medium after 21 days, to 3NM medium aftér 32 days, to 31~' medium after 28 days, to 3NM medium after 2~ days, to 31~' medium after 28 day~, to RM(2~ medium after 29 days, to cubes after 18 days, and to 12" pots after 29 days, One plant was identified as CZ0C22C~. The Rl seeds from this plant were assayed for free pool lysine 2, Line 1007 . . _ Immature embryos were plated on A0, 2N medium . The call~
was transferred and plants regenerated by the following sequence:
to A0.25N ~ medium after 21 days, to A0.5N medium after 25 dayc.
to AlN medium after 30 days, to AlN medium after 25 days, tc HV12A medium after 22 days, to IBM7 medium after 7 days, to RRM medium ~fter 23 days, to cubes after 53 days, and to 12-~ ~ 25 pots after 13 day~. One plant was identified as CZ1418A6, and the ;~ R1 seeds were assayed for free pool Iysine.
12929~
3. Line 1012 lmmature embryos were plated on 3N medium. The callus was transferred and plants regenerated by the following sequence: to 3N medium after 31 days, to 3N medium after 32 days, to 3N
medium after 35 days, to A0.25N medium after 19 days, to A0.25N
medium after 33 days, to A0 . 5N medium after 28 days, to A0 . SN
medium after 29 days, to AlN medium after 29 days, to AlN
medium after 31 days, to AlN medium after 11 days, to R?.11.25 medium after 19 days, to RM2.25 medium after 9 days, to RRM
medium after 29 days, to cubes after 12 days, and to 12" pots after 7 days. One plant was identified as CZ9D15Cl-3.
4. Line 1012 Immature embryos were plated on 3N medium. The callus was transferred and plants regenerated by the following sequence: to 3N medium after 28 days, to 3N medlum after 28 day8~ to 3N3 medium after 30 days, to 3N medium after 37 days, to A0 . 25N
medium after 19 days, to A0.25N medium after 33 days, to A0.5N
medium after 28 days, to A0.5N medium after 29 days, to AlN
medium after 24 days, to AlN medium after 26 days, to AlN
medium after 21 days, to RMl.25 medium after 19 days, to R~2.25 medium after 9 days, to IBM12 medium after 6 days, to RRM
medium after 13 days, to cubes after 23 days, and to 12r pots after 13 day6. One plant wPe identified as CZ9A3BlB9. A second plant identified as CZ9A3BlBll was produced by the same sequence. Rl seeds from these two plants were assayed for free pool ly~e. A third plant was identified as CZ9A3BlA6. The R1 seeds from the third plant were grown and the R2 seeds were analyzed for free pool lysine.
5. Line 1012 ., . . _ Immature embryos were plated on 3N medium. The callus was transferred and plants regenerated by the following sequence; to 1~9Z9~O
3N medium after 26 days, to 3N mediuc: after 30 days, to 3N
medium after 37 days, to A0.25N medium after 19 days, to A0.25N
medium after 33 days, to A2N medium sfter 21 days, to A0.5N
medium after 33 dsys, to A0.5N medium after 16 days, to AlN
medium after 25 days, to AlN medium after 33 days, to RMl.25 medium after 19 da~s, to RM2.25 medium after 9 days, to RRM
medium after 16 days, to cubes after 12 days, and to 12" pots after 22 days. Seven different plants were identified as:
CZ9B9AlAl, CZ9B9AlA6, CZ9B9AlB5, CZ9B9AlB30, CZ9A3A4-3, CZ9A3A4-6, and CZ9C27C2A4. The Rl s~ds from these plants were analyzed for free pool lysine. An additional plant identified as CZ9C27C2A17 was used to produce R2 seeds which were assayed for free pool lysine.
.
6. Line 1010 Immature embr~os were plated on 2N6 ~edium. The callus was trQnsferred and plants regenerated by the followlng sequence: to 2N6 medium after 32 days, to 2N3 mediun~ after 2~ days, to 3NM
medium after 11 d~ys, to 3NM medium after 27 days, to A0.5N
medium after 33 days, to A0.5N medium after 41 days, to AlN
20medium after 28 days, to AlN medium after 26 days, to MAC20 medium after 51 d~s, to MAC15 medium after 32 days, to RMl.5 medium after 44 days, to RM2 . 25 medium after 7 days, to RRMl medium after 33 days, to cube~ after 32 days, and to 12" pots after 4 days. Tv.-o individual plants were identified as CZllC8A2D
25and CZ11C8A2E. Rl seeds from these plants were assayed for free pool lysine.
7. Line 1010 Immature embryos were plated on 2D/Z35 medium. The callus was transferred and plants regenerated by the following sequence:
30to 2N6 medium after 32 days, to 2N3 medium after 27 days, to 3NM
medium after 11 days, to 3NM medium after 27 days, to A0.5N
medium after 33 days, to A0.5N medium after 41 days, to AlN
lZ9Z960 medium after 29 days, to Al~ medium after 23 days, to AlN
medium after 26 days, to MAC20 medium after 51 days, to 2N3 medium after 30 days, to MAC15 medium after 32 days, to RMl.25 medium after 44 days, to RM2.25 medium after 7 days, to cubes after 33 days, and to 12" pots after 22 days. Four different plants were identified as CZllC19AlAlB, CZllC19B2A7, CZllC19B2A8 and CZllC19B2A10. The Rl seeds were analyzed for free pooI Iysine.
8. Line lOlO
Immature embryos were plated on either lOT3S medium (CZllC18) or 2D/Z3S medium (CZllCl9). The callus was transferred and plants regenerated by the following sequence: to 2N6 medium after 32 days, to 2N3 medium after 27 days, to 3NM
medium after 11 days, to 3NM medium after 27 days, to A0.5N
15 medium after 33 d8ys, to A0.5N medium after 41 days, to AlN
medium after 29 days, to A0.5N medium after 23 days, to A0.5N
medium after 26 days, to MAC10 medium after 51 dsys, to 2N3 medium after 30 days, to MAC15 medium after 32 days, to RMl .5 medium after 44 days, to RM2.25 medium after 7 days, to RRMl 20 medium after 33 days, to cubes after 22 days, and to 12" pots after 4 days. Five plants were identified as CZllC18BlCl, CZllC18B2Cl, CZllC19AlBlB, CZllC19AlBlC and CZllC19AlB2B.
Rl seeds were assayed for free pool lysine.
9. Line 1010 Immature embryos were plated on 2N6 medium. The callus was transferred and pIants regenerated by the following sequence: to 2N6 medium after 32 days, to 2N3 medium after 27 days, to 3NM
medium after 11 days, to 3NM medium after 27 day~, to 3NM
medium after 33 days, to AlN medium after 4~ days, to AlN
medium after 32 days, to Al~ medium after 13 day~, to AlN
medium after 35 day~, to MAC20 medium after 52 days, to 2N3 medium after 30 days, to MAC15 medium after 32 days, to P~Ml.2$
lZ9Z960 medium after 44 days, to RM2 . 25 medium after 7 days, to RRM1 medium after 33 days, to cubes after 22 days, and to 12" pots after 14 days. One plant was identified as CZllC9C2, and R
seeds were assayed for free pool lysine.
medium after 11 days, to 3NM medium after 27 day~, to 3NM
medium after 33 days, to AlN medium after 4~ days, to AlN
medium after 32 days, to Al~ medium after 13 day~, to AlN
medium after 35 day~, to MAC20 medium after 52 days, to 2N3 medium after 30 days, to MAC15 medium after 32 days, to P~Ml.2$
lZ9Z960 medium after 44 days, to RM2 . 25 medium after 7 days, to RRM1 medium after 33 days, to cubes after 22 days, and to 12" pots after 14 days. One plant was identified as CZllC9C2, and R
seeds were assayed for free pool lysine.
10. Line 1008 Immature embryos were plated on AO.5N medium. The callus was transferred and plants regenerated by the following sequence:
to AO.lN medium after 36 days, to AO.25N medium after 33 days, to AO.5N medium after 22 days, to AlN medium after 32 days, to 10 AlN medium after 23 days, to AlN medium after 28 days, to Rhll . 25 medium after 20 days, to RM2 . 25 medium after 8 days, to IBM7 medium after 6 days, to RRM medium after 13 days, to cubes after 24 day~, and to 12" pots after 11 da~ s . One plant was identified as CZ13FC1-16, which was used to produce R2 seeds.
15 The R2 BeedB were a88ayed for free pool Iysine.
Following similar procedures as described above, the following plants were also obtained: CZ9D15D2; CZ9C27A1-8; CZ9C27C2A7;
CZ9C27C2A9; CZ9C27C2A18A; CZ9C22AlBlH;CZ9A20C11;
CZgBB48B; CZ9HH2A; CZllC18B2C6; CZllC8A2G; CZ11C20A1;
CZllK8B2C; CZllC19AlB1, and CZ11.
Free Lysine Analysis of Corn Seed A non-destructive single seed assay was used to determine the endogenous free Iysine (endogenous free pool lysine) content 25 ~o~ corn seed of the present invention. In this assay, corn kernels were soaked for 24 hours in sterile water. After soaking, some of the endosperm was removed with a scalpel, being careful not to nick the embryo. The embryo was placed into ~ clean tube 80 that it could be later planted. The pericarp was removed from around 30 the excised endosperm and discarded. The endosperm fragments were placed into a mortar and pulverized thoroughly with a pestle.
lZ5~Z960 The pulverized endosperm was baked to dryness at 100C for one hour. 40 mg of the dried powder was added to a microfuge tube.
A 596 solution of trichloroacetic acid (TCA) was added to each sample to obtain a final concentration of 10 ~1 TCA/mg tissue.
The mixture was mixed thoroughly and allowed to sit for at le~st 30 minutes at room temperature, preferably with continuous shaking. The samples were spun for 10 minutes in a cold room microfuge. 30 1ll of the supernatant were removed and added to a small glass tube or a new microfuge tube. 90 1,l of OPA reaetion - 10 mix were then added and the tube vortex mixed.
The mixture was then immediately subjected to reverse-phase high pressure liquid chromatography to separate the OPA-am~o acid derivatives. A Perkin-Elmer C18 column, 3 ~I particle size, was utilized. 50 ~11 of the sample were injected into the sample loop to insure filling the loop. The OPA-amino acid deriva~es were eluted with a solvent consisting of 3096 acetonitrile and 70~ 50 mM potassium phosphate, pH 6,5, with a flow rate of 1,5 ml/mLn.
The OPA-amino acid derlvativefi were detected by absor~ance at 340 nm. Data collection was ~tarted at 0.1 minute and completet at ~ minutes . The detection threshold was 1. 00 and the minisDu~
peak width was 9.5. The area reject threshold was 2,000 and the vertical scale was 200 m volts. Appropriate control assays ~ere performed to insure complete lysine extraction, OPA derivatizaffon and detection sensitivity.
The free pool lysine content of individual seeds and the average free pool Iysine content of seeds from individual plants are shown in Tables 1 and 2, respectively.
lZ9Z9~i0 Free Pool Lysine Content of Individual Seeds From PIants Produced in Exarnple S
Plant ~ysine (llg/gm Dry Seed Weight) DesignationRl_Seed R2 Seed CZ14I8A6 555.61 --676.00 __ 531.00 __ CZ9C22C* 508.00 --CZ9DlSCl-3514.95 --501.00 __ 800.00 __ CZ9A3BlB9510.00 --CZ9A3BlB11565.00 --CZ9B9AlA6504.08 --718.00 -_ CZ9B9AlB5719.39 --CZ9B9AlB30673.23 --702.00 --735.00 __ 737,00 __ CZ9A3A4-3 534.18 --551.00 __ CZ9A3A4-6 888.77 __ 52g.00 __ 510.00 --653.00 __ CZ9C27C2A4655.00 --CZllC8A2D524.08 --504.06 --CZllC8A2E549.13 --CZllCI9AlAlB 503.76 --523.04 __ 500.07 --::::
TABLE 1 (Cont'd) Lysine (IJ~/gm Dry Seed Weight) Plant DesignationRl Seed R2 Seed CZllC19AlB2B 525.07 --681.55 __ 532.00 __ CZllC19B2A7580.74 --550.09 __ 693.88 --566.32 --CZllC19B2A8524.87 --656.06 --CZllC19B2A10 652.68 --527.08 --664.08 --537. B5 __ CZllC18BlC1540,30 __ 644,73 __ zO 889.64 --CZllC18B2C1566.12 --CZllC19AlBlA 504.84 -- ~
CZllC19AlBlB 520.13 --574.81 __ - CZllClgAlBlC 505.35 --CZllC9C2 502.51 --CZ13FlC1-16 -- 593.05 -- 634.56 C Z9A3B lA6 -- 529.72 CZ9C27C2A17 -- 499.41 -- 521.65 l~9Z960 --3~--Average Free Pool Lysir~e Content of Seeds ~rom Plants Pr~duced in Ex~rnple 5 Average Lysine (~g/g~r Dry Seed Weight) Plant Designation R Seeds R Seeds CZ1418A6 464.09 --CZ9C22C~ 410.g4 --- CZ9C22AlBlH 331.31 --CZ9A20C11 346.52 --CZ9D15CI-3 562.49 --CZ9B9AlAl 441.04 --CZ9B9AlA6 544.69 --CZ9B9AlB5 425.35 _-CZ9B9AlB30 711.81 --CZ9A3A4-3 473.55 --CZ9A3A4-6 645.19 __ CZ~C27C2A4 330.12 --CZ9BB48A 329.49 --CZ9HH2A 333.28 --CZlICBA2D 443.25 --CZllC8A2E 420.13 --CZllC8A2G 401.57 --CZllC19AlAlB 483.21 --CZllC19AlB2B 541.89 --CZllC19B2A7 597.76 --CZllC19B2A8 466.14 --CZllC19B2AlO 595.37 --CZllC20A1 359.49 __ CZllK8B2C 364.87 --CZllC18BlC1 625.78 --C~llC18B2C1 462.99 --CZllC19AlBlB 462.13 --CZllC19AlBlC 377.10 --CZllC19AlBlD 418.19 --lZ9Z96~
TAB LE 2 ( Cont 'd ) Average Lysine (llg/g~T Dry Seed Weight) Plant Desi~ationRl Seeds R2 Seeds CZllC9C2427.50 --CZll 444.70 __ CZ13FlCl-16 -- 540.28 C Z9D15D2 -- 342.70 CZ9C27Al-8 -- 427.10 CZ9A3BlB9 -- 327.14 CZ9A3BlA6 -- 391.46 CZ9C27C2A9 -- 376.47 CZ9C27C2A17 -- 450.35 CZ9C27C2A18A -- 358.12 CZ9C27C2A7 -- 361.25 The lysine content of the starting lines is shown in Table 3 below .
Lysine Content of Starting Lines Lysine Content (~ Dry_Seed Weight) Individual Line Seeds Average 1007 5.00 25.12 12.50 16.30 22.59 69.21 1008 198.00 257.28 267.00 278.00 182.28 361.13 lZ9Z960 TABLE 3 (cont'd) Lysine Content of Starting Lines Lysine Content (l-g/~m Dr~ Seed ~eight) Individual S Line Seeds Avera~e 1010 331.96 281.18 188.72 220.30 304.77 284.62 372. 13 356.51 303. 95 434. 89 188.90 272.85 147,04 320.39 271.05 2a 200.90 257.15 235.79 310.08 308.33 313.22 1012 146.80 142.26 100.40 165.82 ~: ~ 74.74 223,75 ~:
The preceding tables demonstrste the production of malze ~:: seeds which individually have a free pool Iysine conterlt of at le~st 500 1Jg per gram dry tissue weight and of maize plants which lZ9Z96~
produce seeds having an average free pool lysine content of at least 325 IJg per gram dry tissue weight.
Seeds of the lines Crow's 992 (CZ9D15C1-3) and Crow's 1085 (CZ9A3A4-6) were deposited on August 5, 1986 with In Vitro 5 International, Inc., Linthicum, Maryland 21090, and assigned the designated numbers IV1-10114 and IV1-10115, respectively.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to 10 cover any variations, uses or adaptation of the invention following in general, the principles of the invention and including such departures from the present disclosure as come within known and customary practice within the art to which the invention pertains.
to AO.lN medium after 36 days, to AO.25N medium after 33 days, to AO.5N medium after 22 days, to AlN medium after 32 days, to 10 AlN medium after 23 days, to AlN medium after 28 days, to Rhll . 25 medium after 20 days, to RM2 . 25 medium after 8 days, to IBM7 medium after 6 days, to RRM medium after 13 days, to cubes after 24 day~, and to 12" pots after 11 da~ s . One plant was identified as CZ13FC1-16, which was used to produce R2 seeds.
15 The R2 BeedB were a88ayed for free pool Iysine.
Following similar procedures as described above, the following plants were also obtained: CZ9D15D2; CZ9C27A1-8; CZ9C27C2A7;
CZ9C27C2A9; CZ9C27C2A18A; CZ9C22AlBlH;CZ9A20C11;
CZgBB48B; CZ9HH2A; CZllC18B2C6; CZllC8A2G; CZ11C20A1;
CZllK8B2C; CZllC19AlB1, and CZ11.
Free Lysine Analysis of Corn Seed A non-destructive single seed assay was used to determine the endogenous free Iysine (endogenous free pool lysine) content 25 ~o~ corn seed of the present invention. In this assay, corn kernels were soaked for 24 hours in sterile water. After soaking, some of the endosperm was removed with a scalpel, being careful not to nick the embryo. The embryo was placed into ~ clean tube 80 that it could be later planted. The pericarp was removed from around 30 the excised endosperm and discarded. The endosperm fragments were placed into a mortar and pulverized thoroughly with a pestle.
lZ5~Z960 The pulverized endosperm was baked to dryness at 100C for one hour. 40 mg of the dried powder was added to a microfuge tube.
A 596 solution of trichloroacetic acid (TCA) was added to each sample to obtain a final concentration of 10 ~1 TCA/mg tissue.
The mixture was mixed thoroughly and allowed to sit for at le~st 30 minutes at room temperature, preferably with continuous shaking. The samples were spun for 10 minutes in a cold room microfuge. 30 1ll of the supernatant were removed and added to a small glass tube or a new microfuge tube. 90 1,l of OPA reaetion - 10 mix were then added and the tube vortex mixed.
The mixture was then immediately subjected to reverse-phase high pressure liquid chromatography to separate the OPA-am~o acid derivatives. A Perkin-Elmer C18 column, 3 ~I particle size, was utilized. 50 ~11 of the sample were injected into the sample loop to insure filling the loop. The OPA-amino acid deriva~es were eluted with a solvent consisting of 3096 acetonitrile and 70~ 50 mM potassium phosphate, pH 6,5, with a flow rate of 1,5 ml/mLn.
The OPA-amino acid derlvativefi were detected by absor~ance at 340 nm. Data collection was ~tarted at 0.1 minute and completet at ~ minutes . The detection threshold was 1. 00 and the minisDu~
peak width was 9.5. The area reject threshold was 2,000 and the vertical scale was 200 m volts. Appropriate control assays ~ere performed to insure complete lysine extraction, OPA derivatizaffon and detection sensitivity.
The free pool lysine content of individual seeds and the average free pool Iysine content of seeds from individual plants are shown in Tables 1 and 2, respectively.
lZ9Z9~i0 Free Pool Lysine Content of Individual Seeds From PIants Produced in Exarnple S
Plant ~ysine (llg/gm Dry Seed Weight) DesignationRl_Seed R2 Seed CZ14I8A6 555.61 --676.00 __ 531.00 __ CZ9C22C* 508.00 --CZ9DlSCl-3514.95 --501.00 __ 800.00 __ CZ9A3BlB9510.00 --CZ9A3BlB11565.00 --CZ9B9AlA6504.08 --718.00 -_ CZ9B9AlB5719.39 --CZ9B9AlB30673.23 --702.00 --735.00 __ 737,00 __ CZ9A3A4-3 534.18 --551.00 __ CZ9A3A4-6 888.77 __ 52g.00 __ 510.00 --653.00 __ CZ9C27C2A4655.00 --CZllC8A2D524.08 --504.06 --CZllC8A2E549.13 --CZllCI9AlAlB 503.76 --523.04 __ 500.07 --::::
TABLE 1 (Cont'd) Lysine (IJ~/gm Dry Seed Weight) Plant DesignationRl Seed R2 Seed CZllC19AlB2B 525.07 --681.55 __ 532.00 __ CZllC19B2A7580.74 --550.09 __ 693.88 --566.32 --CZllC19B2A8524.87 --656.06 --CZllC19B2A10 652.68 --527.08 --664.08 --537. B5 __ CZllC18BlC1540,30 __ 644,73 __ zO 889.64 --CZllC18B2C1566.12 --CZllC19AlBlA 504.84 -- ~
CZllC19AlBlB 520.13 --574.81 __ - CZllClgAlBlC 505.35 --CZllC9C2 502.51 --CZ13FlC1-16 -- 593.05 -- 634.56 C Z9A3B lA6 -- 529.72 CZ9C27C2A17 -- 499.41 -- 521.65 l~9Z960 --3~--Average Free Pool Lysir~e Content of Seeds ~rom Plants Pr~duced in Ex~rnple 5 Average Lysine (~g/g~r Dry Seed Weight) Plant Designation R Seeds R Seeds CZ1418A6 464.09 --CZ9C22C~ 410.g4 --- CZ9C22AlBlH 331.31 --CZ9A20C11 346.52 --CZ9D15CI-3 562.49 --CZ9B9AlAl 441.04 --CZ9B9AlA6 544.69 --CZ9B9AlB5 425.35 _-CZ9B9AlB30 711.81 --CZ9A3A4-3 473.55 --CZ9A3A4-6 645.19 __ CZ~C27C2A4 330.12 --CZ9BB48A 329.49 --CZ9HH2A 333.28 --CZlICBA2D 443.25 --CZllC8A2E 420.13 --CZllC8A2G 401.57 --CZllC19AlAlB 483.21 --CZllC19AlB2B 541.89 --CZllC19B2A7 597.76 --CZllC19B2A8 466.14 --CZllC19B2AlO 595.37 --CZllC20A1 359.49 __ CZllK8B2C 364.87 --CZllC18BlC1 625.78 --C~llC18B2C1 462.99 --CZllC19AlBlB 462.13 --CZllC19AlBlC 377.10 --CZllC19AlBlD 418.19 --lZ9Z96~
TAB LE 2 ( Cont 'd ) Average Lysine (llg/g~T Dry Seed Weight) Plant Desi~ationRl Seeds R2 Seeds CZllC9C2427.50 --CZll 444.70 __ CZ13FlCl-16 -- 540.28 C Z9D15D2 -- 342.70 CZ9C27Al-8 -- 427.10 CZ9A3BlB9 -- 327.14 CZ9A3BlA6 -- 391.46 CZ9C27C2A9 -- 376.47 CZ9C27C2A17 -- 450.35 CZ9C27C2A18A -- 358.12 CZ9C27C2A7 -- 361.25 The lysine content of the starting lines is shown in Table 3 below .
Lysine Content of Starting Lines Lysine Content (~ Dry_Seed Weight) Individual Line Seeds Average 1007 5.00 25.12 12.50 16.30 22.59 69.21 1008 198.00 257.28 267.00 278.00 182.28 361.13 lZ9Z960 TABLE 3 (cont'd) Lysine Content of Starting Lines Lysine Content (l-g/~m Dr~ Seed ~eight) Individual S Line Seeds Avera~e 1010 331.96 281.18 188.72 220.30 304.77 284.62 372. 13 356.51 303. 95 434. 89 188.90 272.85 147,04 320.39 271.05 2a 200.90 257.15 235.79 310.08 308.33 313.22 1012 146.80 142.26 100.40 165.82 ~: ~ 74.74 223,75 ~:
The preceding tables demonstrste the production of malze ~:: seeds which individually have a free pool Iysine conterlt of at le~st 500 1Jg per gram dry tissue weight and of maize plants which lZ9Z96~
produce seeds having an average free pool lysine content of at least 325 IJg per gram dry tissue weight.
Seeds of the lines Crow's 992 (CZ9D15C1-3) and Crow's 1085 (CZ9A3A4-6) were deposited on August 5, 1986 with In Vitro 5 International, Inc., Linthicum, Maryland 21090, and assigned the designated numbers IV1-10114 and IV1-10115, respectively.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to 10 cover any variations, uses or adaptation of the invention following in general, the principles of the invention and including such departures from the present disclosure as come within known and customary practice within the art to which the invention pertains.
Claims (56)
1. A process for increasing free pool lysine content in maize which comprises:
(a) culturing tissue obtained from a maize plant on a callus induction medium comprising mineral salts, vitamins, sucrose and a hormone to form callus;
(b) subculturing the callus on a selection medium comprising mineral salts, vitamins, sucrose, S-2-aminoethyl-L-cysteine and a hormone to produce callus resistant to S-2-aminoethyl-L-cysteine; and (c) subculturing the resistant callus on regeneration medium comprising mineral salts, vitamins and sucrose to regenerate plants, whereby the free pool lysine content of maize seed is increased to at least about 500 µg per gram dry seed weight or the average free pool lysine content of seeds produced by a maize plant is increased to at least about 325 µg per gram dry seed weight.
(a) culturing tissue obtained from a maize plant on a callus induction medium comprising mineral salts, vitamins, sucrose and a hormone to form callus;
(b) subculturing the callus on a selection medium comprising mineral salts, vitamins, sucrose, S-2-aminoethyl-L-cysteine and a hormone to produce callus resistant to S-2-aminoethyl-L-cysteine; and (c) subculturing the resistant callus on regeneration medium comprising mineral salts, vitamins and sucrose to regenerate plants, whereby the free pool lysine content of maize seed is increased to at least about 500 µg per gram dry seed weight or the average free pool lysine content of seeds produced by a maize plant is increased to at least about 325 µg per gram dry seed weight.
2. The process of claim 1 which further comprises the step of:
(a') subculturing the callus on a maintenance medium comprising mineral salts, vitamins, sucrose and a hormone to maintain the callus before subculturing on the selection medium.
(a') subculturing the callus on a maintenance medium comprising mineral salts, vitamins, sucrose and a hormone to maintain the callus before subculturing on the selection medium.
3. The process of claim 1 wherein the tissue is obtained from immature embryo.
4. The process of claim 2 wherein the tissue is obtained from immature embryo.
5. The process of claim 1 which includes 3-8 subculturing steps on selection medium.
6. The process of claim 2 which includes 2-6 subculturing steps on maintenance medium.
7. The process of claim 6 which includes 3-8 subculturing steps on selection medium.
8. The process of claim 5 wherein the S-2-aminoethyl-L-cysteine concentration in the selection medium is increased stepwise from about 0.1 mM to about 3.0 mM during the subculturing steps.
9. The process of claim 8 wherein the concentration is increased from about 0.5 mM to about 3.0 mM.
10. The process of claim 8 wherein the concentration is increased from about 1.0 mM to about 3.0 mM.
11. The process of claim 7 wherein the S-2-aminoethyl-L-cysteine concentration in the selection medium is increased stepwise from about 0.1 mM to about 3.0 mM during the subculturing steps.
12, The process of claim 11 wherein the concentration is increased from about 0.5 mM to about 3.0 mM.
13. The process of claim 11 wherein the concentration is increased from about 1.0 mM to about 3.0 mM.
14. The process of claim 5 wherein the S-2-aminoethyl-L-cysteine concentration in the selection medium is about 0. 5-0.50 mM
for 1-3 subculturing steps, about 2.0-3.0 mM for 1-2 subculturing steps, and raised stepwise from about 0.5 mM to about 1.0-1.5 mM for the final subculturing steps.
for 1-3 subculturing steps, about 2.0-3.0 mM for 1-2 subculturing steps, and raised stepwise from about 0.5 mM to about 1.0-1.5 mM for the final subculturing steps.
15. The process of claim 7 wherein the S-2-aminoethyl-L-cysteine concentration in the selection medium is about 0.25-0.50 mM
for 1-3 subculturing steps, about 2.0-3.0 mM for 1-2 subculturing steps, and raised stepwise from about 0.5 mM to about 1.0-1.5 mM for the final subculturing steps.
for 1-3 subculturing steps, about 2.0-3.0 mM for 1-2 subculturing steps, and raised stepwise from about 0.5 mM to about 1.0-1.5 mM for the final subculturing steps.
16. The process of claim 8 wherein the S-2-aminoethyl-L-cysteine concentration is dropped to 0 mM for 1 subculturing step after the stepwise incresse and then raised to sbout 1.5-2.5 mM for 1 subculturing step.
17. The process of claim 11 wherein the S-2-aminoethyl-L-cysteine concentrstion is dropped to 0 mM for 1 subculturing step after the stepwise increase and then raised to about 1.5-2.5 mM for 1 subculturing step.
18. The process of claim 1 which includes 2-4 subculturing steps on regeneration medium.
19. The process of claim 2 which includes 2-4 subculturing steps on regeneration medium.
20. The process of claim 18 which includes 3-8 subculturing steps on selection medium.
21. The process of claim 19 which includes 2-6 subculturing steps on maintenance medium.
22. The process of claim 21 which includes 3-8 subculturing steps on selection medium.
23. The process of claim 20 wherein the S-2-aminoethyl-L-cysteine concentration in the selection medium is increased stepwise from about 0 .1 mM to about 3.0 mM during the subculturing steps.
24. The process of claim 23 wherein the concentration is increased from about 0.5 mM to about 3.0 mM.
25. The process of claim 23 wherein the concentration is increased from about 1.0 mM to about 3.0 mM.
26. The process of claim 22 wherein the S-2-sminoethyl-L-cysteine concentration in the selection medium is increased stepwise from about 0.1 mM to about 3.0 mM during the subculturing steps.
27. The process of claim 26 wherein the concentration is increased from about 0.5 mM to about 3.0 mM.
28. The process of claim 27 wherein the concentration is increased from about 1.0 mM to about 3.0 mM.
29. The process of claim 20 wherein the S-2-aminoethyl-L-cysteine concentration in the selection medium is about 0.25-0.50 mM
for 1-3 subculturing steps, about 2.0-3.0 mM for 1-2 subculturing steps, and raised stepwise from about 0.5 mM to about 1.0-1.5 mM for the final subculturing steps.
for 1-3 subculturing steps, about 2.0-3.0 mM for 1-2 subculturing steps, and raised stepwise from about 0.5 mM to about 1.0-1.5 mM for the final subculturing steps.
30. The process of claim 22 wherein the S-2-aminoethyl-L-cysteine concentration in the selection medium is about 0.25-0.50 mM
for 1-3 subculturing steps, about 2.0-3.0 mM for 1-2 subculturing steps, and raised stepwise from about 0.5 mM to about 1.0-1.5 mM for the final subculturing steps.
for 1-3 subculturing steps, about 2.0-3.0 mM for 1-2 subculturing steps, and raised stepwise from about 0.5 mM to about 1.0-1.5 mM for the final subculturing steps.
31. The process of claim 23 wherein the S-2-aminoethyl-L-cysteine concentration is dropped to 0 mM for 1 subculturing step after the stepwise increase and then raised to about 1.5-2.5 mM for 1 subculturing step.
32. The process of claim 26 wherein the S-2-aminoethyl-L-cysteine concentration is dropped to 0 mM for 1 subculturing step after the stepwise increase and then raised to about 1.5-2.5 mM for 1 subculturing step.
33. The process of claim 23 wherein the regeneration medium further comprises S-2-aminoethyl-L-cysteine.
34. The process of claim 26 wherein the regeneration medium further comprises S-2-aminoethyl-L-cysteine.
35. The process of claim 29 wherein the regeneration medium further comprises S-2-aminoethyl-L-cysteine.
36. The process of claim 30 wherein the regeneration medium further comprises S-2-aminoethyl-L-cysteine.
37. The process of claim 31 wherein the regeneration medium further comprises S-2-aminoethyl-L-cysteine.
38. The process of claim 32 wherein the regeneration medium further comprises S-2-aminoethyl-L-cysteine.
39. The process of claim 33 wherein the S-2-aminoethyl-L-cysteine concentration is decreased stepwise from 0.5 mM to 0.1 mM.
40. The process of claim 34 wherein the S-2-aminoethyl-L-cysteine concentration is decreased stepwise from 0.5 mM to 0.1 mM.
41. The process of claim 35 wherein the S-2-aminoethyl-L-cysteine concentration is decreased stepwise from 0.5 mM to 0.1 mM.
42. The process of claim 36 wherein the S-2-aminoethyl-L-cysteine concentration is decreased stepwise from 0.5 mM to 0.1 mM.
43. The process of claim 37 wherein the S-2-aminoethyl-L-cysteine concentration is decreased stepwise from 0.5 mM to 0.1 mM.
44. The process of claim 38 wherein the S-2-aminoethyl-L-cysteine concentration is decreased stepwise from 0.5 mM to 0.1 mM.
45. The process of claim 23 wherein the regeneration medium further comprises one or more cytokinins.
46. The process of claim 26 wherein the regeneration medium further comprises one or more cytokinins.
47. The process of claim 29 wherein the regeneration medium further comprises one or more cytokinins.
48. The process of claim 30 wherein the regeneration medium further comprises one or more cytokinins.
49. The process of claim 31 wherein the regeneration medium further comprises one or more cytokinins.
50. The process of claim 32 wherein the regeneration medium further comprises one or more cytokinins.
51. The process of clsim 33 wherein the regeneration medium further comprises one or more cytokinins.
52. The process of claim 34 wherein the regeneration medium further comprises ona or more cytokinins.
53. The process of claim 35 wherein the regeneration medium further comprises one or more cytokinins.
54. The process of claim 36 wherein the regeneration medium further comprises one or more cytokinins.
55. The process of claim 37 wherein the regeneration medium further comprises one or more cytokinins.
56. The process of claim 38 wherein the regeneration medium further comprises one or more cytokinins.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000553057A CA1292960C (en) | 1987-11-30 | 1987-11-30 | Process for increasing free pool lysine content in maize |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000553057A CA1292960C (en) | 1987-11-30 | 1987-11-30 | Process for increasing free pool lysine content in maize |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1292960C true CA1292960C (en) | 1991-12-10 |
Family
ID=4136965
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000553057A Expired - Fee Related CA1292960C (en) | 1987-11-30 | 1987-11-30 | Process for increasing free pool lysine content in maize |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1292960C (en) |
-
1987
- 1987-11-30 CA CA000553057A patent/CA1292960C/en not_active Expired - Fee Related
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