_??_1992 The Japan Mendel Society Cytologia 57: 339-347 , 1992 Genetic Stability Studies in Regenerated Plants of Allium tuberosum Rottl. ex Spreng. -A Cytological Approach S. Rama Roo, Ruchira Pandey and K. P. S. Chandel National Plant 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 onion (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 garlic-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.