Ploidy research in Clivia

[Tetje]

Mit freundlicher Erlaubnis durch Aart van Voorst:

Ploidy research in Clivia

An update with new results

Aart van Voorst, Netherlands

Summary

After treating seeds of Clivia in vitro and in vivo with colchicine, cytochimerical and tetraploid plants were obtained. Offsets of these plants were tested for ploidy level.
Triploid miniata hybrids were grown to maturity and flowered. Triploid and a tetraploid interspecific hybrid were bred using embryo culture.

Material and methods

In two previous articles methods for converting diploid clivia material into tetraploid were described. The first article (Clivia Five) showed that it is possible to get tetraploid and cytochimerical plants by treating mature embryos of Clivia miniata in vitro with colchicine. The second article (Clivia 6) described a method that made it possible for the amateur breeder to get tetraploid plants without the need of a laboratory.

As previously reported, the first colchicine treatment by me took place in 1995. The resulting polyploid plants were used in different crossings after the first one flowered in 2000. The first crosses were between the polyploid material and orange and yellow diploid miniata hybrids. The aim of these crosses was to get triploid material and to examine if normal seed- forming was possible in the case of diploid-tetraploid crosses in Clivia.

Real advantage for clivia breeding is to be expected when polyploidy is introduced in interspecific hybrids. Combining the genomes of all the Clivia species on a polyploid level could lead to new forms and colors. So, to broaden the tetraploid gene pool crosses were made on a diploid level between several C.miniata yellow hybrids, C.miniata and C.caulescens, C.gardenii, C.nobilis and between C.miniata and the interspecific crosses C.x cyrtanthiflora and a Lotter F2 [(miniata x gardenii) x (nobilis x miniata)]. Both methods of colchicine application were used on the resulting seeds, and to compare these methods in a number of crosses the two methods were used simultaneously.

In addition, seeds of the species C.caulescens, C.gardenii, C.robusta and C.robusta Maxima were used in the project. To investigate what effect polyploidy has on variegation, material from miniata crosses with a variegated mother was included in the programme. On a limited basis germinated seeds from Chinese origin (LOB, variegated, daruma) were also treated with colchicine. Flowcytometer analyses was conducted on offsets of the tetraploid and chimeric plants from the first colchicine treatment in 1995 to test if these offsets were of the same ploidy level as the original plant.

Results


Colchicine treatments


When a colchicine treatment is performed on germinating seeds or embryos, data about the ploidy level can be obtained either by checking the DNA-content, counting the chromosome number, or measuring the stomata or the pollen size (see Clivia Five). The last method requires flowering plants, but this can mean waiting for five years or more. Flowcytometry analyses of the DNA content of the cells are the best method to get results fast. The negative aspect of this method is the relative high cost. Counting the chromosome number in root tips needs a plant that has been growing for some time and has made new roots from that part of the plant that has grown from the colchicine treated meristem. In the layman’s method the original root is not directly in contact with the colchicine (see figure 1.) and may grow on as a diploid root even though the stem meristem has converted into a tetraploid state. I have as yet not had much experience with measuring stomata size, and chimerical tissue may cause problems.




Fig 1. Germinating seeds treated with colchicine using the
Layman’s Method. Material from different crosses on
various colored clay blocks with the stem meristem
covered with colchicine wetted tissue paper and
the root not in direct contact with colchicine


Cross mother x father treated germ.seeds resulting plants
dead 2X Chimeric 4X total
03010 YJK02 x YBW 28 20 8 0 0 8
03012 YJK02 x YPG 39 26 10 3 0 13
sub total 67 46 18 3 0 21
% of treated seeds 69 27 4 0
03018 YJK01 x YPG 44 14 23 6 1 30
03019 YJK01 x YBW 43 14 24 3 2 29
sub total 87 28 47 9 3 59
% of treated seeds 32 55 10 3
Total 154 74 65 12 3 80
% of treated seeds 48 42 8 2 52
YJK Yellow Jaap Keijzer
YBW Yellow Bing Wiese
YPG Yellow Pat Gore

Table 1. Results: colchicine treatment using several yellow C.miniata crosses flowcytometer analyzed. 2X = diploid 4X = tetraploid











Type treated germ.seeds resulting plants
2X Chimeric 4X total
C. robusta Maxima 16 12 0 2 14
% of treated seeds 75 0 13

Table 2. Results: colchicine treatment C.robusta Maxima (flowcytometer analyzed).


A small selection was made of all the colchicine treated material for flowcytometer analysis (Table 1 and 2). Different crosses of related material showed various responses to the colchicine treatment and there also seem to be differences between species. No visual polyploid material was found in either C. caulescens material (61 seeds treated) or C. gardenii material (12 seeds treated). As shown in table 2, C. robusta Maxima on the other hand gave two tetraploid plants.

Results from the comparison of the two methods of colchicine application (vivo and vitro) are not yet available, due to the aforementioned high costs of the flowcytometer analysis.
An advantage of the in vitro method is the higher survival rate of the treated material. In vitro material has more chance to recuperate after the colchicine treatment, being in perfectly conditioned surroundings with good nutrition, although in the layman’s method the still- attached seed also gives enough food for the germinating seedling.

Offsets from polyploid plants

Most plants from the first colchicine treatment in 1995 have formed one or more offsets.
These offsets have been tested for ploidy level. Results show the ploidy level of the mother plants as well as diploid and tetraploid plants (see table 3.)

Plantnumber type Offsets
94001 T=Tetraploid diploid chimeric tetraploid
C=Chimera 2X 2C<X<4C 4X
07 T 1
09 C 1 2
10 C 1 1
12 C 1
13 C 1 1 1
15 C 1
20 C 1
21 C 2
22 C 1
25 C 2 1
26 C 1 1
35 C 1
Total 8 8 5

Table 3. Ploidy level of offsets of colchicine treated material (flowcytometer analyzed)


Seed from diploid x tetraploid crosses

In many plant species, when diploids are crossed with tetraploids, triploid hybrids occur only in very small numbers or not at all. Endosperm failure is the most important reason that seeds don’t develop normally and germination does not take place or leads to an early death of the germinating seed. Removing the embryo from the seed under sterile conditions and placing it on an artificial growing medium in vitro can save the embryo (Embryo Culture). Especially at the start of my polyploidy research all the material from diploid x tetraploid crosses was subject to embryo culture. I did not want to risk losing material because of endosperm failure. Between seven and ten months after pollination the berries of such crosses were surface-sterilized, the seeds were removed and, under sterile conditions, were checked for embryos. Many different stages of development were found for the embryos, also depending on the cross. In some crosses many normal embryos were found, in others only a few or none.



Figure 2. Figure 3.
Different sized embryos from a diploid x tetraploid cross Normal developing embryo from a diploid x tetraploid cross
seen through the bottom of a culture vessel in vitro

Some seeds however did look normal and the question arose whether such seeds would show normal germination in vivo. Figure 4 shows the result of a 2x (=diploid) X 4x (=tetraploid) miniata cross. The seeds were left for natural maturation on the plant. Most seeds are not fully developed, but some are indeed looking normal. In figure 5 some of the normal seeds are shown after chipping and rehydratation. Number one has a normal embryo but will not germinate due to its’ deteriorated endosperm. The embryo could have been saved by embryo culture. Number two looks normal and will germinate the natural way. Number three shows a very big embryo in a small seed: normal germination is doubted, but not impossible.




Figure 4. Seeds from cross 04003: diploid miniata x tetraploid miniata. Figure 5. Geminating seeds from cross 04003
in vivo after chipping and rehydratation..


Flowering of triploid plants

The first triploid flowering took place 4 years after pollination. It is a cross between one of the polyploids as a father and a diploid from the same cross.
This plant as well as all the triploids examined till now, proved to be male sterile. Between the tissue of the shriveled anther some good 2n pollen was found, but there was too little to pollinate with, and it was caught between the anther tissue (see Fig 6.)



Figure 6. Triploid 00002-01. Flower, anthers and colored 2n pollen with anther tissue.

This plant proved to be limited female fertile. One embryo was saved by means of embryo culture after pollinating this triploid with a yellow Vico Gold cross. The resulting plantlet is growing very slowly and the ploidy level has not yet been determined.







Figure 7. YJK01 Figure 8. 94001-20




Figure 9. Selection of triploids from cross 01011.

In 2001 a cross was made between a Jaap Keijzer Yellow as a mother and 94001-20.
Although normal berry development was observed, embryo culture was used after nine and a half months to assure the survival of polyploid embryos.
All the plants that reached maturity (18 in total) proved to be triploid. The triploids came from both groups, normal and abnormal embryos (see table 4.)
The first two plants flowered in 2005, three years after embryo culture. All thirteen plants that have flowered up to now are male sterile. Figure 9 shows different triploids from cross 01011 and figure 10 shows details of plant 01011-15.








Cross number Mother Father # berries # seeds # normal seeds with embryo # abnormal embryos
01011 YJK01 94001-20 7 48 38 14

Table 4. Results from cross 01011





Figure 10. Plant 01011-15

Several crosses were made to get triploid interspecific hybrids. Polyploids were used as father as well as mother. Several triploid interspecifics have been successfully grown from embryos with the aid of embryo culture. There are combinations between C.miniata and C. caulescens, C.miniata and C.gardenii and a combination between C.miniata and C.Xcyrtanthiflora. All these have been flowcytometer analyzed and have the triploid DNA quantity.
The flowcytometer analyses also revealed a pleasant surprise. One of the plants from the caulescens x tetra miniata cross turned out to be tetraploid. Probably an unreduced gamete of caulescens fused with a gamete from the tetraploid miniata.
The material of other colchicine treatments as mentioned above in the section headed “Material and methods” is growing in the greenhouse, but results cannot be given yet. In most of the material slow growing plants with thick leaves are present, which is an indication for polyploidy. Colchicine however also can cause mutations, so thick leaves alone are no proof.

Discussion

Colchicine treatments

Ploidy research in Clivia is an exciting hobby, but it takes many years to get results.
Ten years after the initial colchicine treatment the first triploid flowered. The tetraploid and triploid miniata hybrids are looking good, but don’t stand out yet compared with top quality diploid plants. To get superior polyploid miniata hybrids the gene pool of polyploid material needs to be widened. Material from different sources is being used to contribute to this goal, but the best way to get more different polyploid material is an increased activity on polyploidisation by other Clivia enthusiasts. At this moment it is good to notice that several people are working in this field and this gives hope for the future.
Most of the seeds that I took with me from South Africa after the Clivia Conference in 2002 were small samples of special crosses. I used them in my polyploidisation program, but an individual seed has only a small chance of turning into a polyploid. So only a few plants will be polyploid. Table 1 also shows another aspect that complicates polyploidisation: the two yellow mothers come from the same (genetic) background, but there seem to be differences in reaction between the different crosses. Although a reasonable number of seeds were treated, because of the number of seeds that died after the treatment, not more than an indication can be seen. The treatment of the C.robusta Maxima also gives a different result.

What is most important however is that the Laymans Method as described in Clivia 6 can produce polyploid material.

Offsets from polyploid plants

Colchicine treatment can result in partly polyploid plants. If only a part of the plant has a higher ploidy level, it is possible that the plant can fall back to the original chromosome number and turns diploid again. When partly polyploid plants (cytochimeras) are stable in most cases only one layer of the plant tissue has turned tetraploid. As with most flowering plants (angiosperms), a clivia plant is build from three layers that cover each other like a glove. These layers can be genetically different. The outer layer L1 forms the epidermis of the plant and is only one cell layer thick. The L2 is also likely a single cell layer and forms the next layer and nearly all eggs and pollen are formed from it. The L3, the inner layer, is several cell layers thick. So if a plant becomes a cytochimera, with L2 tetraploid, it will breed like a normal tetraploid. If only L1 gets tetraploid it will breed like a normal diploid.

Offsets of cytochimerical plants can have a different ploidy level compared to the mother plant. The result shown in Table 3 is a good example of this phenomenon. For breeding purposes, working with complete tetraploid plants is preferable, as there is always the danger that a cytochimeric plant can revert to a diploid form. On the other hand, cytochimeric plants may grow faster than complete tetraploids and so be available sooner for further breeding.

Seed from diploid x tetraploid crosses

Although minimal laboratory equipment is required, embryo culture may be too complicated for the amateur breeder to perform. In order to use the potency of polyploidy fully it should be possible to raise triploids from seeds. This has indeed been proven.

In clivia triploids can be raised from seed


Flowering of triploid plants

Triploids are an exciting breeding possibility, when tetraploid material has been obtained. Tetraploidy can sometimes be too high a level of ploidy because of reduced growing speed or flower count. The flowerings of the crosses for triploids, that I made as soon as the first tetraploids flowered, are eagerly anticipated. The first triploid crosses were between miniata material and the results are promising (see fig 6-10). Triploids are by no mains always better than diploids. The same rules that apply for diploid crosses are valid for triploid crosses. The chance that all the desired genes come together in one plant is small, so selection in the progeny is also necessary in triploid material.

There are some characteristics that may directly profit by a higher ploidy level. Polyploidy results in thicker leaf and flower tissue, and may cause a more intense flower colour, This can be especially interesting in breeding e.g. yellow or red. Also, characteristics like leaf width and flower size are positively influenced by polyploidy. To get extremely big flowers or very broadleaf material, the best diploid material for these characteristics should have been used in my colchicine treatments. Such material was not available to me, and my interest in breeding polyploids now focuses primarily on polyploid interspecifics. Triploidy in miniata x caulescens (mxc), miniata x gardenii (mxg) and miniata x Xcyrtanthiflora (mxcyrt) was identified in the Northern spring of 2006. First flowering is expected in 2007. A pleasant surprise was that one of these supposed triploid plants has since been identified as a tetraploid. This tetraploid is most probably a combination of an unreduced gamete of caulescens with tetraploid miniata. This can speed up polyploid interspecific breeding with a couple of years, because the material from the colchicine treated interspecific seeds will not flower for about four years. The tetraploid mxc will also probably be more fertile than the colchicine tetraploids.

Triploid plants are in most cases highly sterile because of the uneven number of genomes in the cell. In forming the gametes - which contain one genome in normal diploid plants, big problems may arise in triploid plants. For example, the triploid Clivias that have flowered so far have all shown shriveled anthers with no free pollen (see fig 6, 9 and 10). Fig 6 shows a microscopic picture of a few very big pollen grains that were still enclosed in the anther tissue. So these plants cannot be used as pollen parents in breeding. The first triploid that flowered was pollinated with pollen from a diploid miniata, and one berry developed. The seed did not develop normally and the embryo had to be saved by means of embryo culture. So there is limited fertility on the mother side in triploid clivia. With this limited fertility it might still be possible to get aneuploid clivia material, i.e, plants with one or more extra chromosomes. This aneuploid material might show special characteristics, if only a limited numbers of genes are present in polyploid number.

Conclusions

Polyploid breeding in Clivia is successful and future developments can contribute to the diversity in Clivia hybrids.
It is possible for the amateur breeder to get tetraploid material using the Layman’s Method and triploid Clivia material can be grown from seeds.