_??_1992 The Japan Mendel Society Cytologia 57: 339-347 , 1992

Genetic Stability Studies in Regenerated of tuberosum Rottl. ex Spreng. -A Cytological Approach

S. Rama Roo, Ruchira Pandey and K. P. S. Chandel

National Tissue Culture Repository, NBPGR Campus, New Delhi-12, India

Accepted January 30, 1992

Nuclear instability of plant cells grown in vitro is very common and has been demonstrated by a number of researchers in wide array of plant materials (D'Amato 1975, Sunderland 1977, Bayliss 1980, Orton 1980). Quite often this instability is manifested in the form of numerical and structural alteration in chromosomes of cultures as well as plants regenerated from them (Kao et al. 1970, McCoy and Bingham 1977, Browers and Orton 1982,Sree Ramulu et al. 1983). The frequency of such aberrations, both structural and numerical, is reported to be very high, especially when the regeneration is through callus (Sacristan 1971, Yamere 1975, Sunderland 1977, Yeoman and Street 1977, Roy 1980) as compared to direct regeneration (Sheridan 1974, Mathur et al. 1987, Sen and Sharma 1991). In both the cases, however, any attempts for clonal multiplication especially when they are made with a prime objective of ex-situ conserva tion should be made with a frequent monitoring and verification of genomic integrity of the cultured material. Cytogenetical analysis, besides biochemical, histological and histochemi cal, is one of the most informative and reliable techniques to ascertain whether any changes occured in the nuclear material during the process of regeneration and organogenesis. Among cytogenetical aspects, karyological studies are of paramount importance as they often pro vide authentic information pertaining to chromosome structure, number and in general their gross morphology (Darlington and La Cour 1976). Meiotic studies on the other hand focus on details of pairing behaviour of chromosomes, recombination frequencies and more impor tantly their disjunctional pattern at anaphase I and II which are not deducible from the mitotic studies (John and Lewis 1965). Allium tuberosum Rottl. ex Spreng. (Familly Alliaceae) is an important wild relative of garden (Jones and Mann 1963) and both diploid (2n=16) and tetraploid (2n=32) cytotypes are known to occur (Mathur and Tandon 1965, Gohil and Koul 1973). The wild genetic resources of this economically important species have been declining in the natural habitat in recent years due to over exploitation by local inhabitants mainly for inflorescences and leaves with -like flavour, and added to this is complete lack of organised cultivation. Necessiated by this fact, efforts were made at National Plant Tissue Culture Repository for in vitro conservation of this important species along with other related types. Subsequently clonal propagation through in vitro shoot proliferation was successfully accomplished (Pandey et al. 1992). In the present investigation, attempts were made to study the genetic stability of regenerated plants in various generations using cytological techniques and this paper deals with a comprehensive account of the results obtained in four generations of A. tuberosum plants raised in vitro.

Material and methods

The standard protocol for the establishment of in vitro regenerated plantlets (Figs. 1, 2) has been discussed elsewhere (Pandey et al. 1992). 340 S. Rama Rao, Ruchira Pandey and K. P. S. Chandel Cytologia 57

Karyological studies were conducted both in vivo (control) and in vitro (regenerated plant lets) materials including four successivepassages designated as P1, P2, P3 and P,. The plantlets of P, culture passage were subsequently established in the field and are designated as P,(F). To ascertain the changes in karyomorphology, studies were made in five regenerants per pas sage of in vitro cultures. A minimum of five slides were prepared from the root tips of each regenerant and on an average four cells from each slide were scored. For meiotic analysis, flower buds of appropriate size were col lected from both control and P4(F) plants simultaneously. Actively growing root tips (ca. 1-2 cm)

were excised and pretreated in double distilled water at 4°C for 24 hrs. After the

pretreatment root tips were fixed over night in 1:3 glacial acetic acid: 95% ethanol.

Later the root tips were stored in 70 ethanol at 10•Ž until squashed. Flower buds were directly fixed in the above men tioned fixative supplemented with a drop of FeCl3 solution for a minimum of 24hr and later stored in 70% ethanol at 10•Ž. For making squash preparation of chromo

somes the materials (root tips/anthers) were washed in 3-4 changes of distilled water and hydrolyzed in IN HCl at 60•Ž for 10min in a water bath. After washing thoroughly, the hydrolyzed materials were transferred to leuco-basic fuchsin solution kept in dark for about 45 min at room temperature. The stained materials were squashed in a drop of 1% aceto-carmine and photomicro

graphs were taken from temporary prepa rations using ORWO MAB photonegative

film. For making slides permanent, the Figs. 1-9. Genetic stability studies in in vitro regenerated plants of Allium tuberosum. 1-2, In cover glasses were removed by liquid nitro vitro propagation. 1, Multiple shoots on B5+0.1 gen freezing technique and dehydrated using mg/l NAA+0.5mg/l Zip in six weeks. 2, Six 1:3, 1:6, 1:9 (glacial acetic acid: 95% month old plants in earthern pots. 3-6, Mitotic ethanol) series followed by two changes in complements. 3, Control (2n=32). 4, P, (2n= 95% ethanol before mounting in D. P. X. 32). 5, P3 (2n=32). 6, P2 (aneusomaty; 2n=26). For analysis of both mitotic and meiotic 7-9, Male meiosis. 7, Control (metaphase 1; 4IV +8II). 8-9, Regenerated plants {P4(F)} metaphase preparations, cells with well flattened and 1; 3IV+10II. clearly countable chromosomes of good morphology, were alone scored.

Observations Mitotic complements Majority of the cells analysed in the root tip cells of control plants exhibited the chromo some number of 32 (Table 1, Fig. 3). A single cell which was aneusomatic (2n=30) was also encountered. The percentage of cells with normal chromosome number was overwhelmingly 1992 Genetic Stability Studies in Regenerated Plants of Allium 341 high in P1,P2, P3, (Table 1, Figs, 4, 5). Ninty six percent cells analysed in P, showed normal chromosome number of 2n=32, while the remaining four percent cells were found to be either aneuploid or polyploid in 3 and I cells respectively. In case of P2 and P3 the proportion of normal cells was calculated to be higher, appreciably (Table 1). The critical analysis of three abnormal cells encountered in P2 revealed the occurrence of aneusomaty in one cell, and the other two cells with some structural changes resulting in visible alteration in chromosome mor phology. On the contrary, all the abnormal cells (3) of P3 plants were recorded as aneuploids. The P4 plants which were analysed prior to their field transfer had about 97% of normal cells, while a small proportion (3%) of cells were observed to be abnormal. Of this, 2% were recorded as aneuploids and 1% with structural alterations (Table 1). The same plants when allowed to grow under field conditions and the secondarly developed roots from them were cytologically analysed, showed more number of normal cells (97.6%). An abnormality ex pressed in the form of aneusomaty (2n=31) in two cells and polyploidy (>4x) in one cell was also recorded. An important finding of the present investigation was that the microsatellited chromosomes were never involved in nuemerical and/or structural alterations and they main tained their integrity in terms of both size and gross morphology. Micronuclei which are reported as primary indicators of nuclear instability and generally frequent in cultured materials, were not encountered in any of the materials studied.

Table 1. Analysis of root tip cells observed at nietaphase in control and regenerated plantlets of A. tuberosum

Associations In control plants, all the 27 pollen mother cells (PMCs) analysed at either diplotene/di akinesis or metaphase I had a mixture of quadrivalents, bivalents and univalents ranging from all univalents to a combination of the above (Table 2, Fig. 7). Multivalents beyond the level of quadrivalents were altogether absent. A characteristic feature observed in the PMCs was the occurrence of an overwhelminglyhigh proportion of quadrivalents as compared to bivalents and univalents. On an average there were 5.77IV+2.6211+3.621 per cell (Table 2). The number of the quadrivalents per cell ranged between 0-8 (Table 4). In as many as 8 out of 27 cells analysed the associations were in the form of all quadrivalents. Among the quadrivalents the ring type have dominated (mean=4.88; range=0-8) the chain types (0.88; 0-4) (Table 2). This led to the occurrence of some cells with only ring quadrivalents, chain types being com pletely absent. Similarly in case of bivalents, ring configurations were more frequently encountered as compared to rod (chain) types. On an average, each cell had 2.18 ring and 0.44 rod bivalents. While as many as 12 ring bivalents could be observed in few cells, the maximum number of rod bivalents recorded was two only. Three out of 27 cells had shown desynapsis and invariably 32 univalents were encountered in all the three cells. 342 S. Rama Rao, Ruchira Pandey and K. P. S. Chandel Cytologia 57

Table 2. Chromosome associations at diplotene/diakinesis and metaphase I

Table 3. Chiasma frequency and terminalization coefficient in control and regenerated plants (P4) of A. tuberosum

Table 4. Frequency of quadrivalents in control and regenerated plants (P4(F)) of A. tuberosum

In case of regenerated plants which were field transferred P4(F), the chromosomal associ ations at diplotene/diakinesis and metaphase I were quite interesting. They exhibited a marked variation in the averages of different associations per cell. Out of 25 PMCs analysed, barring one with 32 univalents, all the others had shown a varying proportion of quadrivalents, biva lents and univalents (Table 2, Figs, 8, 9). On an average each cell had 5.40IV+4.56II+1.4I. In this material also, like control plants, associations beyond the level of quadrivalents were not observed in any of the cells analysed. The relative difference in the proportion of ring and chain/rod types of associations in case of both quadrivalents and bivalents has followed a similar trend as observed in control plants (Table 2). While ring quadrivalents (mean=5.40; range=0-8) out numbered chain types (0.96; 0-2), ring bivalents (3.68; 0-9) were more fre quently encountered as compared to rod bivalents (0.8; 0-5). Another interesting feature ob served in P4(F) plants was the number of quadrivalents per cell ranged between 0-8 (Table 4) and 20% of the cells analysed had all quadrivalents as compared to 29.6% cells in control plants.

Chiasma frequency On an average each PMC in control plant had a chiasma frequency of 28.88 out of which 22.70 were terminalized giving a terminalization coefficient of 0.79 (Table 3). The P,(F) plants, however, had shown a marginal increase in chiasma frequency as well as number of chiasma terminalized. As a result the mean number of chiasma per cell was calculated as 30.56 out of which 25.28 were terminalized giving a terminalization coefficient of 0.84 (Table 3). 1992 Genetic Stability Studies in Regenerated Plants of Allium 343

in the PMCs of control and regenerated plants of A. tuberosum (Table 2. Continued)

Table 5. Proportion of cells with equal/unequal distribution of chromosomes at anaphase I

Chromosome distribution at anaphase I and II 13 out of 20 cells in control plants, observed at anaphase I showed normal distribution (16: 16) (Table 5). The remaining cells exhibited unequal distribution and/or a range of anamolies like laggards/lagging bivalents and late disjunction of chromosomes. The P,(F) differed from the control plants in having more (75 %) cells with normal distribution of chromsomes. The remaining 25 % cells were with either unequal distribution or with some anamolies that are mentioned above (Table 5). Two cells have shown the presence of micronuclei numbering two at anaphase II while some cells showed the occurrence of laggards and/or late disjunction of chromosomes besides normal distribution.

Pollen stainability The control plants showed 75.06% stainable pollens as compared to 73.07% in case of P,(F) plants (Table 5).

Discussion

A critical perusal of literature shows that the stability or lack of it, is an associated phe nomenon of in vitro culture systems and plantlets regenerated from them (D'Amato 1975, Salmia 1975, Sekerka 1977, Roper 1979, Bayliss 1980, Orton 1980, Constantin 1981, McCoy and Phillips 1982, McCoy et al 1982, Papes et al. 1983, Sen and Sharma 1991). A number of workers have demonstrated the instability phenomenon by karyological studies on root tip apices (McCoy and Bingham 1987, Roy 1980,Browers and Orton 1982,Sree Ramulu et al. 1983, Mathur et al. 1987, Sen and Sharma 1991). In contrast only a few reports are available on stability aspects and pairing behaviour of chromosomes during meiosis (Newell et al. 1977, Orton 1980, Edallo et al. 1981, Schenck and Robblen 1982). In the present investigations efforts have been made to study both mitotic as well as meiotic aspects with a prime objective of monitoring the genetic stability of plants regenerated through tissue culture, not only during their culture passages but also after their field establishment. Such systematic and compre hensive analysis in terms of chromosome number, size, morphology, pairing behaviour and their seggregational pattern were rarely reported. At least for various Allium species in general 344 S. Rama Rao, Ruchira Pandey and K. P. S. Chandel Cytologia 57 and Allium tuberosum in particular, no such information is avilable and thus the present in vestigations constitute the first attempt ever made in this direction. As mentioned earlier, dipolid and tetraploid cytotypes of A. tuberosum are known to occur in nature (Gohil and Koul 1971, 1973, Ohno 1964, Mathur and Tandon 1965). However, the occurrence of the polyploid complements (2n=32) in an overwhelming proportion (99.2%) of root tip cells in control plants confirmed that the material used in the present investigations is a tetraploid cytotype. A single exceptional cell with 2n=30 is presumed not to have any be aring either on ploidy level or on the validity of 8 as basic chromosome number of the species, which is widely reported by a number of earlier workers (Kumar and Subarahmaniyam 1987). In polyploid taxa such deviant number of chromosomes in small proportion with no significance whatsoever, are known to exist (Stebbins 1971). The meiotic analysis of this material showed the presence of 8 quadrivalents in 29.6% PMCs studied as compared to 16 reported by Mathur and Tandon (1965). This high proportion of quadrivalents is indicative, if not confirmative, of the autotetraploid nature of the material studied. From Table 1 it can be clearly seen that the regenerants in various culture passages (P1, P2, P3 and P4) and even after their field establishment maintained original chromosome number (2n=32), not withstanding prolonged culture periods. Deviant numbers and karyotypes were encountered, interestingly, only in a small proportion of cells. These observations sug gest that the cultured explants (shoot bases) contained mostly homogeneous cells that are capable of forming adventitious buds and regenerate plants with stable genotypes (Salmia 1975, Papes et al. 1983). Roy (1980) observed high range and frequency of chomosomal abnormalities like aneuploidy, tripolarity and micronuclei etc. in 1-18 months old cultures of a tetraploid cytotype of Allium tuberosum. Contrary to this, such aberrations were very few in number in callus cultures of A. cepa which is a diploid taxa. Based on this, he concluded that the ploidy level of explant material does influence the range and frequencies of chromosomal abnormalities. Papes et al. (1983) also drawn similar conclusions from their studies on Pinus cultures limiting their opinion to only diploid populations. In light of this a distinct feature of the present investigations is that despite tetraploid nature of the explant material, the re generants, helped by the abscence of intervening callus phase, maintained the numerical and structural integrity of chromosomes through successive generations. Abnormalities if any, were limited to a very small proportion of cells. In polyploid species especially autotetraploids, both natural occurring and artificially raised, the reduction in quadrivalent average and corresponding increase in bivalent frequency through successive generations is quite common and has been reported in a number of plant materials viz. Brassica campestris (Swaminathan and Sulbha 1959), Verbena (Arora 1975), Amaranthus (Pal and Khoshoo 1977), Phlox drummondii (Rao et al. 1982) and Tephrosia (Sri vastav 1983). This phenomenon often results in 'diplodization' which in turn helps the plant species especially those bestowed with apomictic behaviour, to adapt to the new physical and physiological environments (Stebbins 1971). Although a one-to-one comparison may be difficultto make still a trend similar to this was observed in P4(F) plants. The data represented in Table 2 with respect to averages of quadrivalents and bivalents in control and P4(F) plants shows a marked drop in the former (from 5.77 to 5.40) and an increase of about 57.5% (from 2.62 in control to 4.56 in P4(F) in case of bivalents. This clearly demonstrates the phenomenon of diplodization, though to a lesser degree, in P4(F) plants. Relatively a smaller proportion of univalents in regenerated plants vis-a-vis control ones appears to indirectly promote the process of diplodization. This is further substantiated by the complete abscence of mul tivalents as well as trivalents which are the only other possible configurations. The P4(F) plants recorded higher frequency of chiasma (30.56) and there by indicated the possibility of more recombinations in genetic makeup of the material. However more number 1992 Genetic Stability Studies in Regenerated Plants of Allium 345 of the chiasma were found to be terminalized giving a greater value of terminalization coef ficient. The corresponding values for control plants were 28.88 and 0.79, respectively (Table 2). A marginal increase in the percentage of cells with equal distribution (16: 16) of chromo somes at anaphase I in P,(F) plants may be directly correlated with increase in bivalent average per cell, which generally tend to disjunct in a more orderly fashion when compared to mul tivalent configurations. This is mainly due to simplicity in the configurations and appropriate orientation of bivalents at metaphase I (Srivastav 1983). Normal meiosis in regenerated plants (P,(F)) with low percentage of pollen stainability indicates that the latter is not due to any chromosomal factor. The involvement of genic and/or physiological factors could, therefore be suspected (Stebbins 1971). The above study conducted on in vitro regenerated plants of A. tuberosum utilizing basal shoot halves aimed at conservation of germplasm and subsequent monitoring of genetic stability of regenerants through several passages as well as after their field establishment. The efficacy of cytological techniques in confirming the nuclear/chromosomal stability in such germplasm has been demonstrated. Variants if any, could be detected through critical karyo logical and meiotic analyses.

Summary Genetic stability, using cytological techniques, has been assessed in regenerated plantlets of Allium tuberosum (Alliaceae) in four culture passages and after their field establishment. Karyological studies of the regenerants indicated that majority (>90%) of the cells had shown normal complements and variants if any, were found to be restricted to a small proportion of cells. Aneusomaty, polyploidy and structural alterations were some of the variations recorded. Meiotic analysis of regenerated plants revealed that by and large they do not differ from the control plants and all the parameters (associations, chiasma frequency, terminalization coef ficient and pollen stainability) showed no significant change when compared to control plants. However, quadrivalent range and frequency showed a considerable reduction in regenerated plants and factors responsible for this were discussed in detail. Efficacy of cytological tech niques in confirming the genetic stability of regenerated plants has been demonstrated.

Acknowledgements

Authors are grateful to Dr. R. S. Rana, Director, National Bureau of Plant Genetic Re sources, New Delhi for providing facilities, and to Department of Biotechnology, Govt. of India, New Delhi for financial assistance. Technical assistance provided by Sh. D. K. Nerwal is also thankfully acknowledged. The authors express thanks to Dr. R. N. Gohil, University of Jammu and NBPGR Srinagar station for providing material.

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