androgenesis in pearl millet

Androgenesis in Pearl Millet I. Analysis of Plants Obtained From Microspore Culture

Due BUI DANG HA and JEAN PERNES

C.N.R.S., Genetique et Physiologie du Developpement des Plantes, F-91190 Gif-sur-Yvette, France

Received June30, 1982 . Accepted September 28, 1982

Summary

The techniques of androgenesis adapted to pearl millet, Pennisetum typhoides, described here have led to the obtention of embryo ids and calluses from all the genotypes used. Plants have been regenerated from 23 D2Bl and from Fl hybrid Massue 9 x Liqui 0' microspore cultures. Two families are obtained by selfing of two doubled haploid plants and compared with the original line 23 D2Bl sown and grown under the same conditions in the greenhouse. They are much less vigourous and have slightly more leaves after one month. Under inductive conditions flowering occurs later. These plants have no seed. Genetic analysis of plants obtained from the Fl hybrid Massue x Ligui concern: a) phenotypic characters: in 160 regenerated plants, the characters «ear shape», «length of main

tiller» and «peduncle length» are variable. However, they all have values close to those of the Ligui parent. This shows that the technique selects against certain type of microspore.

b) biochemical characters: esterases and peroxidases are used as genetic markers. The analysis of these zymograms show some remarkably distorted segregations; the peroxidase P5 is strongly disfavoured and completely unexpected esterase electrophoretic bands are noted.

Key words: Pennisetum typhoides, androgenesis, distorted segregations, peroxidase, esterase.

Introduction

Much careful work has yet to be done in order to establish clearly the potentialities of the use of androgenesis in plant improvement. Maheswari et al. (1980) have enumerated a certain number of problems not yet resolved: the existence of undesirable hereditary modifications induced by androgenesis itself, the possibility of selective pressures favouring only those genotypes responsive to in vitro culture (i. e. capable of easy division and regeneration), and the low productivity of androgenesis (most often only small numbers of plants can be regenerated simultaneously from one plant of a given genotype). In the genus Nicotiana, for which androgenesis is very efficient, Schnell et al. (1980) for N. tabacum, and De Paepe et al. (1977), De Paepe and Pernes (1978) and De Paepe et al. (1981) for N. sylvestris, have shown that androgenesis does not permit the elaboration of doubled haploid lines genetically equivalent to pure lines obtained by successive selfings.

The results presented in this paper show that the regeneration of whole plants by the culture of isolated microspores in pearl millet (Pennisetum typhoides [Burm.] Stapf

Z. Pjlanzenphysiol. Bd. 108. S. 317-327. 1982.

318 Due BUI DANG HA and JEAN PERNES

and Hubb.) 2n = 14, has become successful enough to warrant a systematic evaluation of the potentialities of androgenesis for the improvement of this cereal.

Materials and Methods

The genotypes used as sources of pollen are 23 D2B" a line created by G. W. Burton at Tifton, Georgia; an F 1 hybrid between two lines Massue and Ligui: these lines originate from traditional African cultivars and are maintained by selfing at the laboratory G.P.D.P.; and F2 derived from the Fl by selfing; and a line Massue. These genotypes can be characterized by several well defined genetic markers as shown in Table 1. Electrophoretic patterns of esterase E and peroxidase P isozymes have been used. The techniques of electrophoresis, the identification of the isozymes and their inheritance have been described by Sandmeier et al. (1981). Their sensitivity to photoperiod has been studied by Belliard et al. (1979).

Table 1: Principal characters of the lines Massue and Ligui and their Fl hybrid.

Ligui Massue Fl Genetic control

Peroxidases P3 P3 P3 always present in all lines studied; P5 Ps Ps one locus, one gene, presence dominant

over absence

-EL one gene with two alleles, dimeric Esterases EL EM -EML enzyme, EML is a heterodimer

-EM

seed one or two genes, brown is recessive, colour gray brown gray controlled by maternal nuclear genotype

sensitivity sensitive, partly at least one major gene to photo- neutral quantitative sensitive neutral/sensitive period*) short-day

plant

spike short, globose large, not determined shape cylindrical cylindrical

collar pig- red green red one gene, red dominant over green mentation*)

*) The segregation of these characters is not studied in this article.

The plants are cultivated in the greenhouse of the Phytotron at Gif-sur-Yvette, under controlled conditions: day temperature 28 oe, night temperature 24 oe, 70 % relative humidity, day length 16 h (or 12 h to induce early flowering in plants sensitive to long days), fed with a nutrient solution (Nitsch, 1968).

Pollen isolation and culture

The spikes are detached after they are completely exposed from the flagleaf. At this stage the vacuolization of the uninucleated microspores has not yet begun. The spikes are sterilized in a solution of 7 % calcium hypochlorite for 8-10 min, then rinsed in sterile water. The spikelets are detached from the rachis and planted by sticking their pedicels in gelose medium A contained in 6 cm diameter Petri dishes. The Petri dishes are closed with parafilm and kept in a larger pyrex box. The cultures are placed 30 cm below a red light source (4 Mazdafluor, rouge, TF

Z. Pf/anzenphysiol. Bd. 108. S. 317-327. 1982.

Androgenesis in Pennisetum typha ides 319

40/10 tubes) at 27°C. The anthers emerge from the flowers after about two days. They are collected after a minimum of 12 days of culture. The anthers are then put into liquid medium B in 6 cm Petri dishes. They are cut open in order to release the microspores into the medium; the anther debris is removed using forceps. The cultures are then returned to red light at 27°C. Two or three weeks later, cellular colonies are visible and attain 1-2 mm. If the traditional techniques of grinding anthers and filtering the debris is used, the cells do not divide. They are transferred onto gelose medium C and exposed to white light (Mazdafluor, blanc industrie, TFD 40/BI) 4000-5000 lux, at 27 °C for 5-8 d before being retransferred onto medium D which allows the formation of plantlets.

After a variable length of time, small green points form at the surface of the calluses. These points develop rapidly into whole plantlets. The plantlets are detached from the callus and planted on a solid medium E where they are left for 2 or more weeks before transplantation into pots in the greenhouse. During this period numerous culms and the root system are formed (Fig. 1,2).

The compositions of media A, B, C, D and E are given in Table 2.

B

F

Fig. 1: A, Microspores liberated from the anther after 12-21 days of induction. - B, A microspore dividing after two days of culture. - C, Microspore in a more advanced stage of culture. - D, Formation of a globular embryo. - E, A more advanced embryo. - F, Mitosis of a root tip cell showing n = 7 chromosomes. - Each bar represents 10 /l-m.

Z. Pjlanzenphysial. Bd. 108. S. 317-327. 1982.


Fig. 2: Plant obtained by microspore culture and grown in an Erlenmeyer flask before transfer into a flowerpot.

Table 2: Composition of media used for pearl millet development in androgenesis.

A B C D E

Base medium (Murashige and Skoog, 1962) + + + + + + supplement (1975) Inositol 5 g . 1-1 + + + FeEDTA 5 m\ · \-1 + + + + + pH 5.8 5.8 5.8 5.8 5.8 Activated carbon 2 g·I-1

Yeast extract 500 mg .1-1 + + Saccharose 2% 4% 2% 2% 2% Benzyladenine (B.A.) 300 pg .1-1 100 pg . \-1 Zeatine 50 pg·I-1

I-naphthaleneacetic 500 pg .\-1 1 mg' 1-1 1 mg ' 1-1 200pg·I-1

acid (N.A.A.) (2-4-dichlorophenoxy) 100 pg .1-1 250 pg . 1-1

acetic acid (2,4-D) Glycocoll 2 mg' 1-1 Glutamine 500 mg .\-1 + + + Thiamine 1 mg·\-1 Agar 7% 7% 7% 7%

Z. Pjlanzenphysiol. Ed. 108. S. 317-327. 1982.

Androgenesis in Pennisetum typhoides 321

Two sets of observations are reported here. The first compares the original 23 DzBI line to two families obtained by selfing of two doubled haploid plants Al and Az, obtained by androgenesis from 23 DzBI. The characters measured are: the number of leaves after one month of growth (n), plant height after one month (H1M), and the length and the width of the last adult leaf after one month. The second set of observations concerns an abundant series of more than 100 regenerated plants obtained by androgenesis from the F I hybrid Massue 9 x Ligui (J'. The segregations and recombinations of the characters described in Table 1 and the tillering are directely observed on the regenerated plants. The segregations concern the isozymes P3, Ps and E and seed colour. This family of regenerated plants is also compared by principal component analysis to Ligui and Massue sown under the same conditions, for the following characters: height of principal tiller (H), peduncle length of main spike (P), spike length (LC), spike width (lc), and days of flag leaf appearance after installation in the greenhouse (d). Chromosomes were counted on rdot tip cells of regenerated plants before and after installation in the greenhouse.

Results

Embryoids and calluses have been obtained from all genotypes used. Plandets have been formed from calluses of 23 D2BI (2 plants) and from FI hybrid Massue X Ligui (about 200 plants).

Analysis of regenerated plants from 23 D2BI

Al and A2 were grown from 23 D2BI callus. Their chromosome number in root tips is 2n = 14 but the spikes are mixoploids 2n/4n and have very small numbers of seeds in selfing.

Comparison of 23 D2BI source line and two lines derived from A I and A2 by se/fing: This short study was undertaken in order to determine if it is possible to differentiate an original line from androgenetically derived lines in spite of the absence of qualitative markers. Five out of twenty-two plants of Al and Az seeds were abnormal. The data given in Table 3 concern only the 17 normally developed plants. It shows that

Table 3: Mean values and analysis of variance for three characters of the line 23DB and of two lines (AI and Az) derived from 23DB by androgenesis.

means mean squares F dJ.

Lines 23DB Al Az MSB MSw F2/ 27

height after one month cm 60.1 44.2 45.1 690.7 84.2 9.4*) (HIM)

width of the last adult leaf 22.0 15.2 16.9 126.2 7.8 16.1 *) after one month mm (1)

number of leaves (n) 9.3 9.7 10.6 4.33 0.38 11.5*)

*) Significant at 1 % level.



the plants derived from Al and A2 are smaller and have slightly more leaves after one month: flowering under inductive conditions occurs at least a month later and in some plants floral morphology is quite abnormal. The spikes of all plants analysed have no seed.

Analysis of regenerated plants from the PI hybrid Massue x Ligui

The regenerated plants often present many small culms at the juvenile stage (Fig. 3); later a few of the more robust culms predominate and bear spikes.

Fig. 3: Regenerated plants at the juvenile stage.

Chromosome numbers (root tip mitosis): For 15 plants among the 26 studied, Table 4 shows that the root system is still partially haploid (n = 7) when the plant is transplanted and installed in the greenhouse. Some cells are aneuploid (n = 6). The chromosome number is 2n = 14 for a large proportion of the plants. Mixoploidy (n/2n/3n = 7/14/21) is also observed (Table 4).

Table 4: Chromosome numbers in root tip cells.

Chromosome 7 mixoploids 14 number (once 6) 14 and 7 (once 14

(sometimes 6) and 12)

Number of 4 9 11 plants observed

abnormal numbers 3,7,8, 12

mixoploid total 7/21

26

Aspect of the spike on the most developed tiller, tillering, and flowering: Tillering is systematically more developed in regenerated plants than in the source lines. The principal component analysis (Fig. 4) situates the points representing the regenerated plants in relation to Massue and Ligui, all cultivated under the same conditions.



2 26.31

11 45.87.

Fig. 4: Principal component analysis of plants regenerated from the F 1 hybrid Massue x Ligui. In addition to quantitative measurements (H, P, Lc, Ie and d) the following symbols are used for qualitative notations: • 0 brown, gray for seed colour; 6 0 Ps present absent; If Q 2 or 3 bands for esterase; P '1 only one esterase band, respectively EM or EL· Only 30 plants are described in this analysis; the parents M and L are represented by their means. The first two components describe 72 % of the variation, the first component puts in + value the early, short plants with a thin spike (Ligui type). The second component puts in + value plants with a long spike and long peduncles. We have also drawn the 95 % ellipse of confidence for the regenerated plants.

The regenerated plants are intermediate between the source lines, but a very large heterogeneity is observed among them. The aspect of the main spike only exceptionally resembles Massue.

Table 5 gives the mean values and the genetic variance (V HD) for each character studied.

Fertility: Among the 115 plants that achieved maturity, only 12 didnot yield seed when selfed. Therefore, 89 % of the plants are fertile .

Isoenzyme and coloration markers: Peroxidases: Table 6 shows that the isozyme Ps is strongly disfavoured; if the regenerated plants reflected the theoretical segrega-

Table 5: Variances and means of characters observed in 36 doubled haploid androgenetic plants obtained from an F 1 hybrid Massue (M) x Ligui (L) and in 5 plants of each of the parental lines.

Means Variances

HD L M VHD VL VM

Plant height (H) 62.94 38.01 136.13 327.00 81.45 43.06 Peduncle length (P) 18.592 15.875 16.625 576.18 11.729 8.229 Spike length (Lc) 6.616 6.625 5.950 1.6697 2.563 0.0100 Spike width (Ie) 2.5306 1.900 4.725 0.1473 0.0133 0.0692 Days to flowering (d) 45.38 49.25 84.00 135.60 6.25 24.00

Z. PJlanzenphysiol. Ed. 108. S. 317-327. 1982.


Table 6: Peroxidase zymograms observed in the doubled haploid family obtained from an Fl Massue x Ligui.

Ligui or F 1 type Massue type Abnormal types

Total

Zymogram

P3P5

P3P5 P3P5

P3P5

Number observed

9 103

6 2

120

tion of the pollen of the FJ hybrid, one would expect a Yz P5, Yz 1'5 segregation. Plants without P3 and P5 were also observed.

Esterases: Table 7 shows that the one-band esterase phenotypes normally expected are present only in very small number. By mixing extractions from regenerated plants with extractions from the source lines Massue and Ligui new clearly-defined phenomena can be demonstrated:

1) A one band esterase phenotype that corresponds neither to EL nor to EM' but rather to the heterodimer ELM.

2) A two-band phenotype corresponding to EL and EM' with absence of the heterodimer band ELM.

3) A three-band phenotype, found in the large majority of the regenerated plants, as shown in Table 7. This phenotype does not necessarily correspond to the hybrid phenotype; mixture with an extraction from Massue EM shows four bands. Similar tests, and the observation of the distance between bands in comparison to the con-

L M

Fig. 5: Some esterase zymograms showing the heterogeneity of band patterns in comparison with the Ligui (L) and Massue (M) zymograms.



Table 7: Esterase zymograms observed in the double haploid family obtained from an Fl Massue x Ligui.

Zymogram

EL EM three bands two bands EL and EM one band type ELM

Total

Number observed

8 1

102

8

120

trois, showed that this phenotype is different from the three-band phenotype of F 1

hybrids. It is notable that this three-band phenotype was also observed for plants shown by chromosome count of root tip cells to be n = 7 or mixploids 7/14. Fig. 5 exhibits the diversity of zymograms obtained.

Seed colour (pericarp): Seed colour segregates 80 brown, 12 gray. Nine plants among 92 had complete association of these three characters (gray, P5, EL), which indicates some non-independent distribution of these characters among the regenerated plants (Table 8).

Table 8: Androgenetic plant segregation (Ligui phenotype: gray pericarp color, peroxidase Ps, esterase Ell.

Ligui phenotype L t

Pericarp color gray Peroxidase Ps Esterase EL

12 11 16

80 109 103

L = Ligui phenotype; t = non Ligui.

Discussion - Conclusion

The technique of androgenesis described consists in the induction of supernumerary divisions in precociously isolated pollen grains. Its usefulness is proven by the fact that essentially diploid plants have been regenerated by androgenesis. It would have been easy to produce several hundreds of plants from the Fl pollen; only the nature of the observations and the available space in the greenhouse have limited the number of plants analyzed.

What is the origin of the regenerated plants?

A notable proportion of these plants had haploid cells in their root tips. It would be reasonable to think that they originated from reduced pollen grains and that the progressive doubling of the chromosome number occurs during development. Can we consider that the plants which had 14 chromosomes in all their root-tip cells


326 Duc BUI DANG HA and JEAN PERNES

(42 % of all plants analyzed) originated from non-reduced pollen, as was observed by Wenzel et al. (1976)? The hypothesis of a total non-reduction is to be excluded because these plants do not have a complete F 1 phenotype. Characters like spike form, seed colour, and peroxidase isozymes can present associations that resemble neither the F 1 nor the parental lines. It seems reasonable then to consider that the large majority of the regenerated plants were haploids that spontaneously became diploids more or less quickly.

The segregation of different characters among these regenerated plants obtained by androgenesis presents several interesting features:

1) Numerous recombinations occur and give a clearly diversified series of doubledhaploid plants.

2) The distorsions of some segregations, on the other hand, are spectacular. For peroxidase Ps, .there is a tendency towards the elimination of the Ligui form. For esterase, the analysis is difficult because of the complications introduced by the obtention of three-band phenotypes.

3) Unexpected zymograms have been observed; this is the case for the majority of the esterase zymograms, which are not Ligui, nor Massue, nor Fl type. Neither has the absence of peroxidase P3 ever previously been observed. The study of the self progeny will enable us to find out whether the enzyme phenotypes result from abnormalities induced by in vitro culture.

4) Finally, the two families obtained by selfing diploids obtained androgenetically from 23 D2Bl differed considerably from the source line (weak, late, low fertility) even though they were qualitatively very similar.

All these results demonstrate the necessity for more investigation into genetic changes following androgenesis prior to the establishment of plant breeding programmes.

Acknowledgements

We wish to thank Mrs. M. Heugas and Mrs. E. Nguyen Van for technical help, Mr. B. Fraleigh for translation and Mrs. L. Morice for typing.

References

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z. Pjlanzenphysiol. Bd. 108. S. 317-327. 1982.

androgenesis in pearl millet

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