Techniques in Orchid Cytology

Orchids are generally recognised as cytologically difficult material . The main drawbacks in studying somatic chromosomes are the very slow rate of growth and consequent reduction in the number of dividing cells in the roots and the very compact mataphase plates in the somatic divisions . Added to these are the seasonal activity of the roots and their scarcity in number . The compactness of the somatic metaphase plate is a hindrance to the correct deciphering of the morphology of the chromosomes. So it is necessary that some pretreatment is given to the roots prior to fixation, so that the spindle mechanism is interfered with and the chromosomes are spread out . Colchicine , paradichlorobenzene, 8 -oxyquinoline etc., are some of the chemicals used to achieve this objective .

The fixative most commonly used is Carnoy's fluid (3: 1 of absolute alcohol and glacial acetic acid)or slight modifications of it , as for example 3: 1:1 of absolute alcohol , glacial acetic acid and chloroform. Since orchid chromosomes are generally very small , chromic acid or traces of iron acetate are introduced in order that they take an additional amount of stain.

Carmine and orcein are two of the most popular stains used. Various techniques have been standardised for obtaining good preparations of mitotic and meiotic chromosomes , two of which are described in detail below:

The Mutsuura technique as modified by Ito (Duncan, 1959):

1. Immerse the selected material in 1. 2N HCI for 12 -15 minutes (at 20 degree C. )

2. Rinse in water 30 seconds

3. Treat with chrom-acetic solution (Cro3- 1gm ., glacial acetic acid -3cc. distilled water -100 cc.) for 20 seconds(in case of anther 10-15 seconds).

4. Rinse in water 30 seconds .

5. Treat with ammonia -alcohol solution (30 % alcohol-100 cc. NH4OH-6 drops) for 20 seconds (in case of anther or petal 10-15 seconds).

The microphotopraphs included in plates IX -XVI are from the unpublished Ph.D. thesis (submitted to the University of Kerala. Trivandrum )by Dr. P.Vatsala entitled " Studies on the Cytology and Evolution of Orchidaceae with special reference to Orchids of South India."

Meiotic studies are of pollen mother cells. The magnification of all photographs is approximately x 650.

PLATE IX

Fig.1. Sirhookera Ianceolata Wt. Metaphase I of meiosis. n = 24.

Fig. 2. Nervilia aragona Gaud. Late metaphase of mitosis in stolon tips. 2n= 68.

Fig. 3. Vanilla wightiana Lindl. Metaphase of somatic mitosis. 2n=64.

Fig. 4. Vanilla planifolia Andrews. Somatic mitosis- metaphase . 2n. =32.

Fig. 5. Peristylus stenostachyus Krzl. Second metaphase showing 22 chromosomes at each pole.

Fig. 6. Peristylus stenostachyus Krzl. Meiosis late prophase . Chromosomes show incomplete pairing .

Fig. 7. Peristylus goodyeroides Lindl . Late prophase I of meiosis. Chromosomes show incomplete pairing .

Fig . 8. Peristylus goodyeroides Lindl. Second metaphase showing 23 chromosomes at each pole.

Fig. 9. Habenaria longicornu A. Rich. Metaphase I in meiosis. n=21.

Fig. 10. Habenaria rariflora . A. Rich. Metaphase I in meiosis. n= 21.

Fig. 11. Habenaria longicalcarata A Rich. Meiosis showing 21 bivalents at diakinesis .

Fig. 12. Habenaria crinifera Lindl. Meiotic metaphase I. n=21.

Fig 13. Paphiopedilum drurii Bedd. Mitotic metaphase in root tip cell . 2n= 30.

Fig. 14. Paphiopedilum spicerianum spicerianum Reichenb. f. Mitotic metaphase from root tip. 2n=30.

PLATE X

Fig.15 Nervilia aragona Gaud. Meiosis -Anaphase I showing 34 chromosomes at each pole.

Fig 16. Coelogyne glandulos Lindl. Somatic mitosis . 2n=40.

Fig. 17 . Coelogyne glandulosa Lindl. Meiotic metaphase I . n=20.

Fig. 18 . Coelogyne elata Lindl. Meisosis showing 20 bivalents at metaphase I.

Fig. 19. Pholidota imbricata Lindl. Mitosis in root tip cell. Metaphase 2n= 40.

Fig. 20. Coelogyne elata Lindl. Mitosis in a root tip cell. Metaphase . 2n= 40.

Fig. 21. Coelogyne odoratissima Lindl. Pollen mitosis . Tewnty chromosomes can be seen in each of the four cells of the tetrad.

Fig. 22. Pholidota imbricata Lindl. Meiosis-metaphase I.n=20.

Fig . 23. Coelogyne breviscapa Lindl. Meiosis - Late diakniesis. n=20.

Fig. 24. Coelogyne odoratissima Lindl. Metaphase of mitosis in root cell. 2n =40.

Fig. 25. Coelogyne fuscescens Lindl. -Pollen mitosis .. metaphase. Only three of the four cells of the tetrad are shown . n=20.

Fig 26. Coelogybe corrugata Wt. Pollen mitosis -metaphase . n=20.

Fig 27. Coelogyne flavida Wall. Meiosis -metaphase I. n= 20.

Fig. 28. Arundina chinensis Bl. Mitosis in root cell-metaphase. 2n=40.

PLATE XI

Fig . 29 . Coelogyne nervosa . S. Rich. Mitosis in root cell. 2n = 40 .

Fig . 30 . Coelogyne ovalis Lindl. Meisosis I showing 40 bivalents .

Fig 31. Arundina bambusifolia Lindl. Anaphase I of meiosis . n. =20 .

Fig 32. Arundina bambusifolia Lindl. Root cell mitosis -metaphase 2n =40 .

Fig 33. Epidendrum radicans Pavon. Late metaphase I of meiosis . n =40.

Fig . 34. Epidendrum xanthinum Lindl. Pollen mitosis metaphase showing 30 chromosomes in each of the four cells of the tetrad.

Fig. 35. Epidendrum radicans Pavon. Anaphase I of meiosis . n=40.

Fig .36. Epidendrum xanthinum Lindl. Meiotic metaphase I showing irregularities such as clumping of bivalents etc.

Fig. 37. Dendrobium macrostachyum Lindl. Root tip cell mitosis -metaphase 2n = 38 .

Fig 38. Dendrobium macrostachyum Lindl. Meiosis metaphase I. n. =19.

Fig. 39. Dendrobium Dendrobium pierardi Roxb. Mitosis in root tip cell. 2n = 38.

Fig. 40. Dendrobium pierardi Roxb. Meiosis metaphase I. n=19.

Fig 41. Dendrobium pierardi Roxb. Meiosis anaphase I . n= 19 .

Fig 42. Dendrobium tortile Lindl. Meiosis anaphase I. n=19.

PLATE XII

Fig . 43. Dendrobium tortile Lindl. -Metaphase I in two pollen mother cells. in =19.

Fig . 44 .Dendrobium chrysotoxum Lindl. Root tip cell mitosis . 2n =38.

Fig. 45. Dendrobium chrysotoxum Lindl. Meiosis -metaphase I . n=19.

Fig . 46. Dendrobium chrysanthum Wall. Metaphase in root tip cell. en=38.

Fig 47. Dendrobium moschatum Wall. Var Cupreum Metaphase II of meiosis . n=19.

Fig . 48. Dendrobium grande Hook. Mitosis in a young leaf cell- metaphase. 2n=38.

Fig . 49. Dendrobium herbaceum Lindl. Metaphase I of meiosis showing 20 bivalents and a small fragment .

Fig . 50. Dendrobium coelogyne Reichenb. Mitos in a root tip cell showing 40 normal chromosomes and four smaller ones super numeraries?)

Fig 51. Dendrobium barbatulum Lindl. Metaphase I of meiosis showing 20 bivalents .

Fig 52. Dendrobium fytcheanum Batem. Meiosis-metaphase I. n=20. Some of the bivalents show a tendency to associate in twos and threes.

Fig 53. Dendrobium Chlorops Lindl. Meiotic metaphase I showing 20 bivalents .

Fig 54. Desmotrichum fimbriatum Bl. Meiosis -metaphase I .n=19.

Fig . 55 . Polystachya wightii Reichenb.f. Meiotic anaphase I. n=20.

Fig . 56. Podochilus malabaricus Wt. Meiosis -metaphase I. n=20.

Fig . 57. Spathoglottis plicata Bl. Meiosis in the pink-flowered variety -metaphase I. n=20.

Fig . 58. Calanthe vestita Lindl. Somatic mitosis from a root tip cell. 2n=42.

Fig. 59. Calanthe veratrifolia R. Br. Metaphase from a root tip cell. 2n=40.

Fig. 60. Spathoglottis plicata Bl. Meiosis showing irregularities at metaphase I. Bivalents forming rings and chains.

Fig. 61. Calanthe veratifolia R. Br. Pollen mitosis in tetrad. each cell showing 20 chromosomes at metaphase .

Fig. 62. Calanthe masuca Lindl. Variety I. Meataphase I of meiosis . Grouping of bivalents is frequently noted.

Fig. 63. Spathoglottis plicata Bl. Var alba Ridl. Meiosis showing 20 bivalents.

Fig. 64. Calanthe vestita Lindl. Metaphase II in Meiosis . n=21.

Fig 65. Calanthe veratrifolia R. Br. Anaphase I of meiosis . n=20.

Fig 66. Phaius veratrifolius Lindl. Meiosis-metaphase I.n=25.

Fig 67. Calanthe veratrifolia R.Br. Late diakinesis of meiosis . n=20.

Fig 68. Calanthe masuca Lindl. Variety I. Mitosis in a root tip cell. 2n=40.

PLATE XIV

Fig. 69. Calanthe masuca Lindl. Meiotic anaphase I. n=20.

Fig 70. Calanthe masuca Lindl. Pollen mitosis showing 20 chromosomes in each cell.

Fig . 71. Calanthe masuca Lindl. Metaphase of pollen mitosis in a diad cell showing the doubled number of chromosomes (40).

Fig. 72. Calanthe masuca Lindl. Variety II. Mitosis in a root tip cell-metaphase . 3n=60.

Fig . 73. Calanthe masuca Lindl. Triploid variety. Early anaphase I of meiosis .

Fig 74 & 75 Calanthe masuca Lindl. Meiosis in triploid variety showing trivalents .

Fig. 76 . Late prophase II of meiosis in the above variety showing unequal separation leading to 35 chromosomes in one nucleus and 25 chromosomes in the other .

Fig . 77. Bulbophyllum neilgherrense Wt. Variety I. Meiotic metaphase I. n=19.

Fig . 78 . Bulbophyllum neilgherrense Wt. Variety II. Meiotic metaphase I. n=19.

PLATE XV

Fig 79. Bulbophyllum tremulum Wt. Meiotic metaphase I. n=19.

Fig 80. Bulbophyllum albidum Hook. Mitosis in a root cell-metaphase. 2n=38.

Fig 81. Geodorum densiflorum Schltr. Mitosis in a root tip cell. 2n=52.

Fig 82. Geodorum densiflorum Schltr. Meiosis-metaphase I. n=26.

Fig 83. Cymbidium aloifolium Sw. Mitosis in a root tip cell. 2n= 40 .

Fig 84. Grammatophyllum tigrinum Blume. Meiosis -metaphase I. n=20.

Fig 85. Peristeria elata Hook. Meiosis-metaphase I .n=20.

Fig 86. Peristeria elata Hook. Meiosis -Anaphase I showing 20 chromosomes at each pole.

Fig 87. Aerides radicosum A. Rich. Meiotic metaphase I. n=19.

Fig 88. Rhynchostylis retusa Bl. Meiosis -metaphase I. n=19.

Fig 89. Oncidium luridum Lindl. Mitosis in root cell. 2n=26.

Fig 90. Saccolabium pulchellum Fischer. Three pollen mother cells each showing meiotic metaphase I.n. =19.

Fig 91. Cottonia macrostachya Wt. Mitosis in root cell . 2n=38.

PLATE XVI

Fig. 93. Oncidium luridum Lindl. Meiotic metaphase I. n=13.

Fig 94. Aerides ringens Fischer . Meiotic metaphase I. n=19.

Fig 95. Sarcanthus peninsularis Dalz. Mitosis in root cell. 2n=38.

Fig 96. Acampe wightiana Lindl. Meiotic metaphase I. n=19.

Fig 97. Aerides odoratum Lour. Meiotic anaphase I. n=19.

Fig 98. Acampe wightiana Lindl. Mitosis in root cell . 2n. =38.

Fig 99. Sarcanthus peninsularis Dalz. Meiotic metaphase I. n=19.

Fig 100. Luisia tenuifolia Bl. Mitosis in root cell . 2n =38.

Fig 101. Vanda parviflora Lindl. Mitosis in root cell. 2n=38.

Fig. 102. Vanda parviflora Lindl. Meiotic metaphase I. n=19.

Fig 103. Vanda tesselata Hook. Mitosis -metaphase . 2n=38.

Fig 104. Vanda hookeriana Reichenb.f. Meiotic anaphase I. 19 chromosomes in each pole.

Fig . 105. Vandatessellata Hook. Meiotic metaphase I. n=38.

Fig 106. Vanda spathulata Spreng. Meiotic metaphase I. n=38.

Fig 107. Vanda spathulata Spreng . Meiotic anaphase I showing regular separation of 38 chromosomes to each pole.

6. Rinse in water 30 seconds .

7. Place the material on a slide and drop over it acetocarmine solution, iron being added by way of a steel needle . After covering with a cover glass , press and slip it slightly in order to spread the material . Next eaise the end of the cover glass to squash thoroughly.

8. Pass the inverted slide six or seven times through a weak spirit flame.

9. Press cover glass again slightly .

The staining technique which has been standardise at the Hawaii Agricultural Experiment Station by Dr. Kamemoto and his associates makes use of the orcein stain and involves the following procedure:

Three types of fixatives are recommended by Kamemoto , Tanaka and Kosaki (1966) -3:1 of absolute alcohol and acetic acid , 1: 1: 1 of absolute alcohol ,acetic acid and chloroform and just 45% acetic acid .