Utilization of Wild Arachis Species at ICRISAT

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A. K. Singh, D. C. Sastri and J. P. Moss*

One of the possibilities for increasing the yield leaf spots. These were A. cardenasii Krap. and of groundnut, particularly in the Semi-Arid Greg., nomen n u d u m , A. chacoense Krap. and Tropics, is breeding varieties w i t h resistance to Greg., nomen n u d u m , and Arachis species Coll. pests and diseases. S o me progress has been HLK 410 which were reported as i m m u n e to made in this field, but the improvements that Cercosporidium personatum, highly resistant can be made by breeders are limited by the to Cercospora arachidicola, and resistant to availability of genes within A. hypogaea. Collec- both (Abdou 1966; Sharief 1972; Abdou et al. tions of wild species f r o m South America have 1974; Hammons, personal communication). made available a wider range of genes, espe- The groundnut cytogenetics program at cially genes for disease resistance. The richness ICRISAT was initiated in April 1978 with the of Arachis germplasm collection offers a great object of making the fullest possible use of the opportunity for anyone interested in the im- genus Arachis. Cooperation with the Genetic provement of this crop (Bunting et al. 1974, Resources Unit, pathologists, entomologists, Simpson 1976; Smartt et a I. 1978a, b; Gregory and microbiologists has increased the number and Gregory 1979). of wild species at ICRISAT and our knowledge However the c y t o t a x o n o m y of the genus of the desirable genes which they contain. Arachis is such that it is difficult for a breeder to Cytogenetic analysis provides information to use wild species in groundnut improvement improve the efficiency of incorporation of wild (Gregory and Gregory 1979; Moss 1980; Stalker species genes into A. hypogaea. Thetechniques 1980). The t w o major constraints to utilization have been described by Singh et al. (1980). of w i l d species are differences in ploidy level In addition to A. hypogaea, section Arachis and incompatibility between some wild species contains one other tetraploid. A. monticola, and and A. hypogaea. The small size of chromo- several diploid species. All these species are somes of Arachis and the difficulty experienced cross compatible with A. hypogaea (Smartt and by some workers in making good cytological Gregory 1967; Stalker 1980). Nine diploid preparations has deterred many people from species and the two tetraploids have been attempting cytogenetic techniques in the im- studied in detail and a chromosome with a sec- provement of Arachis, despite their successful ondary constriction and a small satellite seen as application to many other crop plants, espe- chromosome 3 in A. villosa, A. correntina, A. cially w h e a t and t o b a c c o . The g r o u n d n u t chacoense, and Arachis species Coll. No. 10038, cytogenetics program at ICRISAT has attemp- and a chromosome with a secondary constric- ted to overcome these difficulties, and to pro- tion and a large satellite seen in A. batizocoi and duce interspecific hybrids and to manipulate in A. duranensis as chromosome 2, in Arachis the ploidy level to produce tetraploid lines in- species 338280 as chromosome 6 and in A corporating desirable characters which can cardenasii as chromosome 9. Chromosomes then be utilized by breeders in the improvement with secondary constrictions had only been of groundnut. reported previously in A. batizocoi. The small The program on utilization of wild species in pair of chromosomes in A. cardenasii are larger Arachis was initiated at Reading University than in the remaining species but still smaller (U.K.) w i t h three species which were known to than the smallest chromosomes of A. batizocoi. cross w i t h A. hypogaea and were resistant to A. monticola and A. hypogaea are close karyomorphologically, though A. monticola has t w o pairs of chromosomes w i t h secondary constrictions whereas A. hypogaea has only * Cytogeneticists, Groundnut Improvement Pro- g r a m , ICRISAT. one, and the chromosome with a secondary

82 constriction in A. cardenasii is comparable to These basic studies are designed to assist us one of those of A. monticola and that of A. in the main objective of the cytogenetics unit, hypogaea. i.e., the utilization of the wild species for the 2 Mahalanobis D analysis and canonical improvement of groundnuts. The two go hand analysis, using the arm ratio of each of the ten in hand because m a n y of t h e plants pro- chromosomes of the diploid taxa investigated, duced in the course of utilization of wild species resulted in t w o clusters within these. A. are analyzed in detail, and plants which are batizocoi is the only species in one of these analyzed are then used in crossing programs. clusters; there are eight taxa in the other cluster, The t w o main thrusts of the subprogram are the which can be further subdivided (Fig. 1). use of compatible species to transfer currently All the species of the section Arachis are available genes, and the study of the barriers to cross-compatible, and differences in crossabi- hybridization and the means of breaking them lity are too small to be satistically significant. All to make the whole gene pool within Arachis the available nine diploid taxa have been cros- available to breeders in the future. sed in all possible combinations and a large n u m b e r of F1 hybrids have been analysed B r e e d i n g i n C o m p a t i b l e S p e c i e s cytologically. Results from these studies sub- stantiate our grouping of the diploid species. The incorporation of genes from wild species The F1 hybrids resulting f r o m the cross of t w o involves t h e transfer of one or m o r e w i l d species belonging to the t w o different clusters species genomes to a hybrid where they can show a high frequency of univalents and a high undergo recombination with A. hypogaea pollen sterility, while F1 hybrids between t w o genomes, and subsequent transfer of the de- species of the same cluster show a low fre- sired gene or genes into A. hypogaea with the quency of univalents and a low pollen sterility. elimination of all undesirable characters from the wild species. Five routes have been adopted to achieve these objectives (Fig. 2). D2 distances among species of section Arachis Triploid Route

A. villosa Smartt and Gregory (1967), Moss and Spielman (1976), Raman (1976), and Moss (1977) have all produced hexaploids by chromosome doubling A. correntina in a triploid hybrid. As early as 1976, ICRISAT received hexaploids from the program at Read- A. chacoense ing University. These hexaploids combined the genomes of A. cardenasii, A. chacoense and A. sp 263133 Arachis species No. 338280 with A. hypogaea, (SL) and were screened for leaf spot resistance at

2 ICRISAT, which is mainly infested by late leaf A. sp 263133 D =0.99 A. batizocoi (LL) spot (C. personatum). Resistant plants were selected from each type of hexaploid, and have A. sp 338280 been backcrossed to different cultivars of A. hypogaea (Moss 1980). These progenies are A. duranensis n o w in the fourth generation of backcrossing and tetraploid and near tetraploid plants are 2 D = 0 . 3 7 9 - 0 . 8 8 3 being screened for disease resistance. Many hexaploids were also resistant to rust. There is A. cardenasii no correlation between leaf spot or rust resistance and defoliation in hexaploids derived Cluster I Cluster II from resistant wild species, as some hexaploids Average intracluster D2 = 0.335 susceptible to disease do not defoliate. Con- versely, some hexaploids with few small le- Figure 1. Cluster diagram. sions suffer severe defoliation (Moss et al.

83 GENOME TRANSFER GENE TRANSFER

(1a) 4X Cult. 3X C h r o m o s o m e 6X doubling 2X Wild (1b) 4 X Cult. 3X 5 X - 6 X 2X Wild (2) 2X Wild C h r o m o s o m e 4X Reciprocal doubling 4X backcrossing 4X Cult. with (3) 2 x W i l d A. hypogaea 2X Chromosome. 4X doubling 2X Wild (4) 4X Wild 4X Cult. 4X 4X Cult.

Figure 2. Utilization of diploid and tetraploid wild species.

1979). The fertility of t h e h e x a p l o i d s and Autotetraploid Route backcrosses ranges f r o m nil, in some sterile but vegetatively vigorous plants, to highly produc- The production of an autotetraploid from a dip- tive. Some plants produce many pegs per node loid wild species enables all hybridization and and pegs per plant, but few pods, whilst others genome transfer to be done at the tetraploid have good reproductive efficiency. level, and increases the dosage of wild species Crosses between A. hypogaea and five dip- genes. Autotetraploids have been produced in loid species have produced 92 pods (Table 1). seven taxa (Table 2). Of these only A. batizocoi Five triploids have been established f r o m spr- (4x) has produced seed; many plants of the outs and the remaining seed will be used to others remained sterile and eventually died. produce hexaploids. However, three autotetraploids were success- The artificial induction of polyploidy in a trip- fully used as male parents in crosses with A. loid to restore fertility, can be difficult and time hypogaea (Table 3). The resultant progenies are consuming, so the development of a technique morphologically similar to A. hypogaea, but whereby large numbers of cuttings of the sterile cytologically unstable, and sterile. A number of triploid can be treated has increased the pro- generations of selfing of the autotetraploids will duction of hexaploids (Spielman and Moss increase the frequency of balanced gametes, 1976; Nigam et al. 1978). Triploids can produce and the likelihood of fertile hybrids with A. fertile gametes through the formation of a re- hypogaea. Hybrids will be backcrossed to A. stitution nucleus and by segregation giving 2n hypogaea to restore fertility whilst selecting de- or near 2n gametes. Studies of Anaphase I of sirable recombinants. meiosis show that 30, 20 and hyper-20 c h r o m o - some cells occurred; triploids have produced seed in the field at ICRISAT. Amphiploid Route This process may be environmental and/or Chromosomedoubling in a hybrid between two genotype specific; for instance high tempera- diploid wild species produces an amphidiploid ture in India may be one of the reasons for the which combines the t w o wild species, which is formation of unreduced gametes, and the trip- t h e s a m e ploidy level as tetraploid A. hypogaea, loids differ in the frequency w i t h which they and increases the number of genomes in the produce seed. This latter may be due to the hybrid and therefore the number of possible different w i l d species used, or an effect of the recombinants. If the t w o wild species used are different A. hypogaea genotypes w h i c h are the ancestors of A. hypogaea, the amphidiploid known to affect the amount of pairing in hexa- will be a synthetic A. hypogaea, and this may be ploids (Spielman et al. 1979). the most promising tetraploid derivative of the

84 T a b l e 1. Crossability b e t w e e n A. Hypogaea and diploid species of section Arac his.

No. of No./% N o . / % Cross Pollinations of pegs of pods

A. hypogaea x A. correntina 63 24/38 22*/35 A. hypogaea x A. batizocoi 64 20/31 16/25 A. hypogaea x A. villosa 87 38/44 26/30 A. hypogaea x A. duranensis 58 19/33 14*/24 A. hypogaea x A. sp 338280 44 15/34 14/32

Total 316 116/37 92/29

* Sprouts produced.

T a b l e 2. P r o d u c t i o n of a u t o t e t r a p l o i d s f r o m w i l d diploid Arachis species.

Seedlings Plants 2x 4x Species treated survived plants plants

A. villosa 17 14 11 4 A. correntina 18 16 10 2 A. chacoense 5 1 A. sp 338280 19 12 3 3 A. sp 263133 8 5 4 1 A. duranensis 7 6 2 2 A. cardenasii 15 14 1 3 A. batizocoi 26 21 11 10

T a b l e 3. Crossability b e t w e e n A. hypogaea and autotetraploids of section Arachis.

No. of No./% No./% Cross pollinations of pegs of pods

A. hypogaea x A. sp 338280 (4x) 132 16/12 13*/10 A. hypogaea x A. villosa (4x) 139 30/22 20*/14 A. hypogaea x A. sp 263133 (4x) 113 19/17 17/15

Total 384 65/17 50/13

* Sprouts p r o d u c e d .

wild species with regard to the genetic im- and cytologically; their behavior is similar to provement of A. hypogaea. Intracluster crosses the progenies obtained through the autotetrap- are much more successful than intercluster loid route. The amphiploids involving three crosses (Fig. 3). species are the most fertile. Fifty-one amphiploids have been raised from 17 different cross combinations of wild species, Use of Tetraploid Wild Species including A. batizocoi (Table 4), and eight different combinations have been successfully cros- A. monticola has been crossed with A. sed with A. hypogaea (Table 5). The resultant hypogaea and fully fertile hybrids have been progenies are being analyzed morphologically produced.

85 A. villosa This means that there is a good probability of gene transfer through chromosome pairing in A. correntina these hybrids and backcrosses producing new gene combinations in progenies, enabling A. chacoense selection of the desired plants.

A. sp 338280 Induced Translocation A. sp 263133 (LL) 27/16 A. batizocoi Such chromosome pairing may not always A. sp 263133 (SL) occur, either because of the incorporation of a nonhomologous genome, or because the gene- A. duranensis tic background of the hybrid prevents pairing of homologous genomes. In such cases translocation of chromosome segments has to be in- A. cardenasii duced through mutagenesis.

Total no. of pollinations 5348 Total no. of pegs 1453 Barriers to Hybridization Total no. of pods 901 and Means of Breaking Them

Figure 3. Percentages of peglpod production A hypogaea has not been successfully and in interspecific crosses in section repeatably crossed with any species outside Arachis. section Arachis (Gregory and Gregory 1979). Many species in other sections have potential as gene sources in groundnut im- G e n e Transfer provement (Moss 1980). Tetraploid wild species are found in section Rhizomatosae (Gregory and Gregory 1979) and these species are of By Chromosome Pairing special interest as they are immune to many Our cytological analysis of the diploid wild pathogens. Several attempts have been made species, F1 hybrids and triploids resulting from by many people over the years to achieve an crosses of wild diploid species with A intersectional hybrid but these have not been hypogaea has indicated that there is chromo- successful (Gregory and Gregory 1979). Recent some homology or homoeology among the advances in the knowledge of the physiology genomes of all the wild diploid species studied and development of pollen, factors involved in and between these genomes and A hypogaea. pollination and advances in the technology of

Table 4. Number of amphiploids established in section Arachis. sp 263133 chacoense correntina duranensis A. cardenasii A A A A sp 338280 A. A. chacoense x

A villosa 6 2 A. correntina 4 1 5 A sp 338280 2 2 A. duranensis 1 1 3 5 A. sp 263133 2 3 A. batizocoi 2 1 8

86 Table 5. Crossabllity batween A. hypogaea and amphlploids.

No. of No./% No./% Cross pollinations of pegs of pods

A. hypogaea X [A. correntina X 160 26/16 22*/14 [A chacoense x A. cardenasii)]

A. hypogaea

X [A. sp. 338280 X 164 42/26 32*720 (A. chacoense x A. cardenasii)]

A. hypogaea X 199 62/31 42*721 {A. correntina x A. villosa)

A, hypogaea X 135 15/11 15*/11 {A. villosa x A. sp 338280)

A. hypogaea X 96 9/9 7»/7 (A duranensis x A. chacoense)

A. hypogaea X 29 8/28 7/24 (A batizocoi x A. villosa)

A. hypogaea X 18 7/39 5*/28 (A batizocoi x A. duranensis)

A. hypogaea X 10 7/70 3*/30 [A. batizocoi x A. chacoense)

Total 811 176/22 133/16

* Sprouts produced.

tissue culture have increased the possibility of were smooth with small callose patches distri- producing hybrids between species which were buted evenly along the lengths of the pollen previously considered to be genetically isolated tubes. In the incompatibly pollinated pistils, (Heslop Harrison 1978; Vasil 1978, 1980; however, the callose depositions along the Shivanna et al. 1979; Sala et al. 1980). pollen tube were uneven and in larger quan- In June 1979 a project was initiated to investi- tities indicating a retarded growth of the tubes. gate the barriers to intersectional hybridization. However, a small frequency of incompatible Fluorescent microscopic comparison of the pollinations induced peg initiation and elonga- compatibly and incompatibly pollinated pistils tion, though these usually dried and degener- showed that in the former, the pollen tubes ated before they penetrated the soil.

87 A number of techniques have been tested for repeatable results. We were able to induce their efficiency to overcome such incompatibili- normal embryogeny in one ovule culture up to ty. Plant growth hormones applied to the ovary the cotyledonary stage of the embryo by using were found to increase the frequency of peg an aqueous peg extract in the medium. formation in incompatible crosses. The initial While excising the embryos from seed for trials, using cytokinin at 10-6M applied to cotton culture, the cotyledons were also cultured. We webs wrapped round the ovaries, were fol- observed that the end of the cotyledon proximal lowed by trials using four concentrations of to the embryo is a highly embryogenic tissue kinetin and three of benzylamino purine, as well which gives rise to roots, shoots, embryoids or as auxins and gibberellic acid. The effects of whole plants depending upon the hormonal these treatments are shown in Table 6. Kinetin, balance in the medium. We have also been naphthaleneacetic acid and gibberellic acid have trying to regenerate plants from leaflet seg- all significantly increased the pegging percen- ments and have been able to induce roots but tage. not shoots or embryos, although we have tried Some of these pegs have been left to form four different basic media, White (1943), pods in the soil. Others have been excised from Murashige and Skoog (1962), Gamborg et al. the plants for aseptic culture of the tip of the (1972) and Kao and Mickayluk (1975), with a peg, or the ovule, or the embryo. We have so far range of auxins and kinetins, as well as supple- been able to culture immature embryos suc- menting with coconut milk, casein hydrolysate, cessfully into seedlings using Murashige and yeast extract, malt extract or gibberellic acid. Skookg's 1962 medium. Our attempts to culture pegs according to Ziv and Zamsky (1975), or Conclusion ovules according to Martin (1970), even from compatible crosses, have not given satisfactory Considerable progress has been made with a

Table 6. Effact of soma plant growth hormones on pegging after pollination of tetraploid species of section Anchls with Arachis sp PI No. 276233 of section Rhizomatotae.

Arachis monticola A. hypogaea var . Robut33-1 No. of Pegs formed No. of Pegs formed Cross Treatment pollinations (%) pollinations (%)

Compatible None 201 34.33 156 34.62 (self) Incompatible None 314 17.20 147 15.65 (control) Incompatible Kinetin, 10-4 M 21 14.29 " Kinetin, 10-5 M 90 15.56 82 25.00 " Kinetin, 10-6 M 75 18.67 55 25.46 It Kinetin, 10-7 M 95 14.75 87 40.23 " Benzyl Adenine, 10-5M 134 22.39 " Benzyl Adenine, 10-6M 191 20.42 " Benzyl Adenine, 10-7M 98 19.39 " Indole Acetic Acid 107 13.08 53 24.53 " (25 ppm) » Napthalene Acetic Acid 129 17.83 52 42.31 (25 ppm) " 2, 4-Dlchloro Phenoxy- 73 17.81 18 22.22 acetlc Acid (25 ppm) * Gibberellic Acid 37 48.65 (25 ppm) " Kinetin 70-4M + 89 24.72 Indole Acetic Acid (25 ppm)

88 number of different ways of utilizing wild species. ceedings of the International Legume Conference, The number of plants produced, and the range Kew, England, 1978. of variation they show, indicate that there is good potential for transferring desirable charac- Moss, J. P., and SPIELMAN, I. V. 1976. Interspecific ters f r o m wild species. Although the wild hybridization in Arachis. Proceedings of the Ameri- can Peanut Research and Education Association species were originally considered solely as 8: 88 (Abs.). sources of disease resistance, the progenies from interspecific crosses have potential as a Moss, J. P., SINGH, A. K., BURGE, A. P., and BRADLEY, S. means of expanding the gene pool of Arachis 1979. Wild species in the improvement of with respect to a number of other desirable groundnuts. 1, Disease reaction of hexaploids. Pro- characters. ceedings of the American Peanut Research and Results of attempts to break the barriers to Education Association 11: 55 (Abs.). hybridization hold promise for utilization of characters f r o m species outside section MURASHIGE, T., and SKOOG., F. 1962. A revised medium for rapid growth and bioassays with to- Arachis. bacco tissue cultures. Physiologic Plantarum 15: 473-497.

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